Novel core 2 beta-1, 6-N-acetylglycosaminyltransferas E gene

ABSTRACT

The invention provides novel core 2 β-1,6-N-acetylglycosaminyltransferase nucleic acids, and polypeptides encoded by the nucleic acids. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists, and methods for treating disorders associated with the enzyme.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is related to U.S. patent application Ser. No.09/495,913 filed Feb. 2, 2000, which claims the benefit of ProvisionalPatent Application No. 60/118,674 filed Feb. 3, 1999, the teachings ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to novel core 2β-1,6-N-acetylglycosaminyltransferase nucleic acid molecules,polypeptides encoded by such nucleic acid molecules, and uses of thenucleic acid molecules and polypeptides.

BACKGROUND OF THE INVENTION

[0003] The enzyme UDP-GlcNAc:Gal[β] 1,3GalNAc-R (GlcNAc to GalNAc) [β]1,6-N-acetylglucosaminyltransferase (i.e. core 2β-1,6-N-acetylglycosaminyltransferase) converts core 1 (i.e. Gal[β]1,3GalNAc[α]-O) to core 2 structures (i.e.Gal[β]1,3[GlcNAc[β]1,6]GalNAc[α]-O in the O-linked glycan biosynthesispathway (Williams and Schachter, 1980 J. Biol. Chem 255:11247, 1980 andSchachter H. and Brockhausen, I, In: Allen, H. J. and Kisailus, E. C.(eds) Glycoconjugates. Composition, Structure, and Function. MarcelDekker, New York, pp 263-332). Core 2 GlcNAc-T activity is important inthe extension of O-linked sugars with poly(N-acetyllactosamine) (i.e.repeating Gal [β] 1-4GIcNAc [β] 1-3). These structures have beenassociated with malignant transformation (Yousefi et al, 1991) andproliferative activation of lymphocytes (Higgins et al, 1991), theyaffect cellular adhesion (Zhu and Laine, 1985; Laferte and Dennis,1988), and they may act as ligands for mammalian lectins (Merkle andCummings, 1988)

[0004] Synthesis of branched, complex core 2-based O-linked structureshas been found to be controlled by the relative levels of core 2GlcNAc-T and [α]-2,3 sialyl-T (Whitehouse et al, 1997) which compete forthe same core 1 acceptor substrate. Therefore, core 2 is a key enzyme inthe modulation of cell-cell interactions through glycosylation of targetmolecules. For example, glycosylation of PSGL-1 modulated by core 2GlcNAc-T has been found to be a critical step for binding to P-selectin(Kumar et al, 1996; Li et al, 1996).

[0005] Expression of Core 2 GlcNAc-T in diabetic heart has also beenassociated with a stress-response and myocardial hypertrophy (Nishio Y.et al, J. Clin Invest October 1995; 96(4): 1759-67). Diabetes andhyperglycemia induces core 2 GlcNAc-T gene expression specifically incardiac myocytes of rats.

[0006] GalNAcαR prevents core 2 synthesis by blocking one enzyme earlierin the O-linked pathway, and it reduces invasion and metastasis. Asomatic mutation that prevents UDP-Gal transport into the Golgi blockingO- and N-linked extensions including core 2 structures causes a moresevere attenuation of metastasis than a block in either pathway alone,suggesting both O-linked core 2 and N-linked branched oligosaccharidescontribute to the malignant phenotype. Most recently, it wasdemonstrated that an increased expression of core 2 GlcNAc-T incolorectal cancer cells is closely correlated with the progression ofthedisease (Shimodaira K., at al 97, Cancer Res.).

[0007] The identification of new core 2 GlcNAc-transferases and nucleicacids encoding the enzymes satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention, andtreatment of disorders mediated by the enzymes including cancer,cardiovascular disorders, and inflammatory disorders.

[0008] The citation of any reference herein is not an admission thatsuch reference is available as prior art to the instant invention.

SUMMARY OF THE INVENTION

[0009] The present inventors have identified novel core2-β1,6-N-acetylglycosaminyltransferase nucleic acid molecules, andpolypeptides encoded by such nucleic acid molecules. The nucleic acidmolecules are herein designated “core2b GlcNAc-T” or “core2b GlcNAc-T”,and the polypeptides are herein designated “Core 2b”, “Core 2bGlcNAc-T”, or “Core 2b GlcNAc-T Polypeptide”. The core 2b GlcNAc-Tnucleic acid molecules were found to be primarily expressed in thegastrointestinal tract.

[0010] Broadly stated the present invention contemplates an isolatedCore 2b GlcNAc-T nucleic acid molecule encoding a polypeptide of theinvention, including mRNAs, DNAs, cDNAs, genomic DNAs, PNAs, as well asantisense analogs and biologically, diagnostically, prophylactically,clinically or therapeutically useful variants or fragments thereof, andcompositions comprising same.

[0011] The invention also contemplates an isolated Core 2b GlcNAc-Tpolypeptide encoded by a nucleic acid molecule of the invention atruncation, an analog, an allelic or species variation thereof, or ahomolog of a polypeptide of the invention or a truncation thereof.(Truncations, analogs, allelic or species variations, and homologs arecollectively referred to herein as “Core 2b GlcNAc-T RelatedPolypeptides”). The polypeptide comprises cytosolic, transmembrane, andcatalytic regions.

[0012] The nucleic acid molecules of the invention permit identificationof untranslated nucleic acid sequences or regulatory sequences thatspecifically promote expression of genes operatively linked to thepromoter regions. Identification and use of such promoter sequences areparticularly desirable in instances, such as gene transfer or genetherapy, which may specifically require heterologous gene expression ina limited environment. The invention therefore contemplates a nucleicacid molecule comprising a non-coding sequence such as a 5′ and/or 3″sequence, preferably a non-coding sequence of core2b GlcNAc-T,preferably a sequence as shown in SEQ. ID. NO. 5 or 6.

[0013] The nucleic acid molecules which encode for the mature core2bGlcNAc-T polypeptide (may include only the coding sequence for themature polypeptide (SEQ ID NO. 1, 3, and 19); the coding sequence forthe mature polypeptide and additional coding sequences (e.g. leader orsecretory sequences, proprotein sequences); the coding sequence for themature polypeptide (and optionally additional coding sequence) andnon-coding sequence, such as introns or non-coding sequence 5′ and/or 3′of the coding sequence of the mature polypeptide (e.g. SEQ ID NO. 5 and6).

[0014] Therefore, the term “nucleic acid molecule encoding apolypeptide” encompasses a nucleic acid molecule which includes onlycoding sequence for the polypeptide as well as a nucleic acid moleculewhich includes additional coding and/or non-coding sequences.

[0015] The nucleic acid molecules of the invention may be inserted intoan appropriate vector, and the vector may contain the necessary elementsfor the transcription and translation of an inserted coding sequence.Accordingly, vectors may be constructed which comprise a nucleic acidmolecule of the invention, and where appropriate one or moretranscription and translation elements linked to the nucleic acidmolecule.

[0016] Vectors are contemplated within the scope of the invention whichcomprise regulatory sequences of the invention, as well as chimeric geneconstructs wherein a regulatory sequence of the invention is operablylinked to a heterologous nucleic acid, and a transcription terminationsignal.

[0017] A vector can be used to transform host cells to express a Core 2bGlcNAc-T Polypeptide or Core 2b GlcNAc-T Related Polypeptide, or aheterologous polypeptide (i.e. a polypeptide not naturally expressed inthe host cell). Therefore, the invention further provides host cellscontaining a vector of the invention. The invention also contemplatestransgenic non-human mammals whose germ cells and somatic cells containa vector comprising a nucleic acid molecule of the invention inparticular one that encodes an analog of Core 2b GlcNAc-T, or atruncation of Core 2b GlcNAc-T.

[0018] The polypeptides of the invention may be obtained as an isolatefrom natural cell sources, but they are preferably produced byrecombinant procedures. In one aspect the invention provides a methodfor preparing a Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-TRelated Polypeptide utilizing the purified and isolated nucleic acidmolecules of the invention. In an embodiment a method for preparing aCore 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T Related Polypeptideis provided comprising:

[0019] (a) transferring a vector of the invention comprising a nucleicacid sequence encoding a Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide, into a host cell;

[0020] (b) selecting transformed host cells from untransformed hostcells;

[0021] (c) culturing a selected transformed host cell under conditionswhich allow expression of the Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide; and

[0022] (d) isolating the Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide.

[0023] The invention further broadly contemplates a recombinant Core 2bGlcNAc-T Polypeptide, or a Core 2b GlcNAc-T Related Polypeptide obtainedusing a method of the invention.

[0024] A Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T RelatedPolypeptide of the invention may be conjugated with other molecules,such as polypeptides, to prepare fusion polypeptides or chimericpolypeptides. This may be accomplished, for example, by the synthesis ofN-terminal or C-terminal fusion polypeptides.

[0025] The invention further contemplates antibodies having specificityagainst an epitope of a Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide of the invention. A purified antibody whichspecifically binds to a Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide of the invention is provided. Antibodiesmay be labeled with a detectable substance and used to detectpolypeptides of the invention in biological samples, tissues, and cells.

[0026] The invention also permits the construction of nucleotide probesthat are unique to nucleic acid molecules of the invention and/or topolypeptides of the invention. Therefore, the invention also relates toa probe comprising a sequence encoding a polypeptide of the invention,or a portion (i.e. fragment) thereof. The probe may be labeled, forexample, with a detectable substance and it may be used to select from amixture of nucleic acid molecules a nucleic acid molecule of theinvention including nucleic acid molecules coding for a polypeptidewhich displays one or more of the properties of a polypeptide of theinvention.

[0027] In accordance with an aspect of the invention there is provided amethod of, and products for (i.e. kits), diagnosing and monitoringconditions mediated by core 2b GlcNAc-transferases by determining thepresence of nucleic acid molecules and polypeptides of the invention

[0028] Still further the invention provides a method for evaluating atest compound for its ability to modulate the biological activity of aCore 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T Related Polypeptideof the invention. For example, a substance which inhibits or enhancesthe catalytic activity of a Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide may be evaluated. “Modulate” refers to achange or an alteration in the biological activity of a polypeptide ofthe invention. Modulation may be an increase or a decrease in activity,a change in characteristics, or any other change in the biological,functional, or immunological properties of the polypeptide.

[0029] Compounds which modulate the biological activity of a polypeptideof the invention may also be identified using the methods of theinvention by comparing the pattern and level of expression of a nucleicacid molecule or polypeptide of the invention in biological samples,tissues and cells, in the presence, and in the absence of the compounds.

[0030] In an embodiment of the invention a method is provided forscreening a compound for effectiveness as an antagonist of a polypeptideof the invention, comprising the steps of

[0031] a) contacting a sample containing said polypeptide with acompound, under conditions wherein antagonist activity of saidpolypeptide can be detected, and

[0032] b) detecting antagonist activity in the sample.

[0033] Methods are also contemplated that identify compounds orsubstances (e.g. polypeptides) which interact with core2b regulatorysequences (e.g. promoter sequences, enhancer sequences, negativemodulator sequences).

[0034] The nucleic acid molecules, polypeptides, and substances andcompounds identified using the methods of the invention, may be used tomodulate the biological activity of a Core 2b GlcNAc-T Polypeptide, or aCore 2b GlcNAc-T Related Polypeptide of the invention, and they may beused in the treatment of conditions mediated by core 2bGlcNAc-Transferases such as inflammatory disorders, liver disorders,gastrointestinal disorders, diabetes, and proliferative diseases such ascancer. Accordingly, the nucleic acid molecules, polypeptides,substances and compounds may be formulated into compositions foradministration to individuals suffering from one or more of theseconditions. Therefore, the present invention also relates to acomposition comprising one or more of a polypeptide, nucleic acidmolecule, or substance or compound identified using the methods of theinvention, and a pharmaceutically acceptable carrier, excipient ordiluent. A method for treating or preventing these conditions is alsoprovided comprising administering to a patient in need thereof, acomposition of the invention.

[0035] The present invention in another aspect provides means necessaryfor production of gene-based therapies directed at the gastrointestinaltract. These therapeutic agents may take the form of polynucleotidescomprising all or a portion of a nucleic acid molecule of the inventioncomprising a regulatory sequence of core 2b GlcNAc-T placed inappropriate vectors or delivered to target cells in more direct ways.

[0036] Having provided a novel Core 2b GlcNAc-T Polypeptide, and nucleicacids encoding same, the invention accordingly further provides methodsfor preparing oligosaccharides e.g. two or more saccharides includingsLe^(x) antigens. In specific embodiments, the invention relates to amethod for preparing an oligosaccharide comprising contacting a reactionmixture comprising an activated GlcNAc, and an acceptor in the presenceof a Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T RelatedPolypeptide of the invention.

[0037] In accordance with a further aspect of the invention, there areprovided processes for utilizing polypeptides or nucleic acid molecules,for in vitro purposes related to scientific research, synthesis of DNA,and manufacture of vectors.

[0038] These and other aspects, features, and advantages of the presentinvention should be apparent to those skilled in the art from thefollowing drawings and detailed description.

DESCRIPTION OF THE DRAWINGS

[0039] The invention will be better understood with reference to thedrawings in which:

[0040]FIG. 1 shows an alignment of a nucleic acid molecule of theinvention (SEQ. ID. NO.1) and human core 2 DNA (SEQ. ID. NO.13);

[0041]FIG. 2 shows an alignment of a polypeptide of the invention andhuman core 2 polypeptide (Accession No. 544360; SEQ. ID. NO.14);

[0042]FIG. 3 is a blot showing expression of core2b GlcNAc-T mRNA inhuman tissues;

[0043]FIG. 4 is a. blot showing expression of core2b GlcNAc-T MRNA ingastrointestinal tissues;

[0044]FIG. 5 is a blot showing expression of core2b GlcNAc-T in normal(N) and tumor (T) tissues; and

[0045]FIG. 6 is a blot of an RT PCR analysis of core 2b GlcNAc-T insamples from normal colon and liver and from patients with colon cancerand liver metastasis.

DETAILED DESCRIPTION OF THE INVENTION

[0046] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See for example, Sambrook, Fritsch, & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y); DNA Cloning: APractical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization B. D. Hames & S. J. Higgins eds. (1985); Transcription andTranslation B. D. Hames & S. J. Higgins eds (1984); Animal Cell CultureR. I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press,(1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984).

[0047] Nucleic Acid Molecules of the Invention

[0048] As hereinbefore mentioned, the invention provides isolated Core2b GlcNAc-T nucleic acid molecules. The term “isolated” refers to anucleic acid (or polypeptide) removed from its natural environment,purified or separated, or substantially free of cellular material orculture medium when produced by recombinant DNA techniques, or chemicalreactants, or other chemicals when chemically synthesized. Preferably,an isolated nucleic acid is at least 60% free, more preferably at least75% free, and most preferably at least 90 to 99% free from othercomponents with which they are naturally associated. The term “nucleicacid” is intended to include modified or unmodified DNA, RNA, includingmRNAs, DNAs, cDNAs, and genomic DNAs, or a mixed polymer, and can beeither single-stranded, double-stranded or triple-stranded. For example,a nucleic acid sequence may be a single-stranded or double-stranded DNA,DNA that is a mixture of single-and double-stranded regions, or single-,double- and triple-stranded regions, single- and double-stranded RNA,RNA that may be single-stranded, or more typically, double-stranded, ortriple-stranded, or a mixture of regions comprising RNA or DNA, or bothRNA and DNA. The strands in such regions may be from the same moleculeor from different molecules. The DNAs or RNAs may contain one or moremodified bases. For example, the DNAs or RNAs may have backbonesmodified for stability or for other reasons. A nucleic acid sequenceincludes an oligonucleotide, nucleotide, or polynucleotide. The term“nucleic acid molecule” and in particular DNA or RNA, refers only to theprimary and secondary structure and it does not limit it to anyparticular tertiary forms.

[0049] In an embodiment of the invention an isolated nucleic acidmolecule is contemplated which comprises:

[0050] (i) a nucleic acid sequence encoding a polypeptide havingsubstantial sequence identity with the amino acid sequence of SEQ. ID.NO. 2, 4, 7, 8, 9, or 20;

[0051] (ii) a nucleic acid sequence complementary to (i);

[0052] (iii) a nucleic acid sequence differing from any of (i) or (ii)in codon sequences due to the degeneracy of the genetic code;

[0053] (iv) a nucleic acid sequence comprising at least 10, 15, 18,preferably at least 20 nucleotides capable of hybridizing to a nucleicacid sequence of SEQ. ID. NO. 1, 3, 10, 11, 12, or 19 or to a degenerateform thereof;

[0054] (v) a nucleic acid sequence encoding a truncation, an analog, anallelic or species variation of a polypeptide comprising the amino acidsequence of SEQ. ID. NO. 2, 4, 7, 8, 9, or 20; or

[0055] (vi) a fragment, or allelic or species variation of (i), (ii) or(iii)

[0056] In a specific embodiment, the isolated nucleic acid moleculecomprises:

[0057] (i) a nucleic acid sequence having substantial sequence identityor sequence similarity with a nucleic acid sequence shown in SEQ. ID.NO. 1,3, 10, 11, 12, or 19;

[0058] (ii) a nucleic acid sequence complementary to (i), preferablycomplementary to the full nucleic acid sequence shown in SEQ. ID. NO. 1,3, 10, 11, 12, or 19;

[0059] (iii) a nucleic acid sequence differing from any of the nucleicacid sequences of (i) or (ii) in codon sequences due to the degeneracyof the genetic code; or

[0060] (iv) a fragment, or allelic or species variation of (i), (ii) or(iii).

[0061] The term “complementary” refers to the natural binding of nucleicacid molecules under permissive salt and temperature conditions bybase-pairing. For example, the sequence “A-G-T” binds to thecomplementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, in which only some of thenucleic acids bind, or it may be complete when total complementarityexists between the single stranded molecules.

[0062] In a preferred embodiment the isolated nucleic acid comprises anucleic acid sequence encoding a polypeptide having an amino acidsequence of SEQ. ID. NO. 2, 4, or 20, or comprises the nucleic acidsequence of SEQ. ID. NO. 1, 3, 10, 11, 12, or 19 wherein T can also beU.

[0063] The terms “sequence similarity” or “sequence identity” refer tothe relationship between two or more amino acid or nucleic acidsequences, determined by comparing the sequences, which relationship isgenerally known as “homology”. Identity in the art also means the degreeof sequence relatedness between amino acid or nucleic acid sequences, asthe case may be, as determined by the match between strings of suchsequences. Both identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W. ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G. eds. HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, New York, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds. M. Stockton Press, New York,1991). While there are a number of existing methods to measure identityand similarity between two amino acid sequences or two nucleic acidsequences, both terms are well known to the skilled artisan (SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, New York,1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds. M.Stockton Press, New York, 1991; and Carillo, H., and Lipman, D. SIAM J.Applied Math., 48:1073, 1988). Preferred methods for determiningidentity are designed to give the largest match between the sequencestested. Methods to determine identity are codified in computer programs.Preferred computer program methods for determining identity andsimilarity between two sequences include but are not limited to the GCGprogram package (Devereux, J. et al, Nucleic Acids Research 12(1): 387,1984), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol.215:403, 1990). Identity or similarity may also be determined using thealignment algorithm of Dayhoff et al [Methods in Enzymology 91: 524-545(1983)].

[0064] Preferably, the nucleic acids of the present invention havesubstantial sequence identity using the preferred computer programscited herein, for example greater than 50%, 60%, 70%, 75%, 80%, 85%, or90% identity; more preferably at least 95%, 96% 97%, 98%, or 99%sequence identity to the sequence shown in SEQ. ID. NO. 1, 3, 10, 11,12, or 19.

[0065] Isolated nucleic acids encoding a Core 2b GlcNAc-T Polypeptideand comprising a sequence that differs from the nucleic acid sequence ofSEQ. ID. NO. 1, 3, 10, 11, 12, or 19 due to degeneracy in the geneticcode are also within the scope of the invention. Such nucleic acidsencode equivalent polypeptides but differ in sequence from the sequenceof SEQ. ID. NO. 1, 3, 10, 11, 12, or 19 due to degeneracy in the geneticcode. As one example, DNA sequence polymorphisms within core2b GlcNAc-Tmay result in silent mutations that do not affect the amino acidsequence. Variations in one or more nucleotides may exist amongindividuals within a population due to natural allelic variation. Anyand all such nucleic acid variations are within the scope of theinvention. DNA sequence polymorphisms may also occur which lead tochanges in the amino acid sequence of Core 2b GlcNAc-T Polypeptide.These amino acid polymorphisms are also within the scope of the presentinvention. In addition, species variations i.e. variations in nucleotidesequence naturally occurring among different species, are within thescope of the invention.

[0066] Another aspect of the invention provides a nucleic acid moleculewhich hybridizes under selective conditions, (e.g. high stringencyconditions), to a nucleic acid which comprises a sequence which encodesa Core 2b GlcNAc-T Polypeptide of the invention. Preferably the sequenceencodes the amino acid sequence of SEQ. ID. NO. 2, 4, or 20 andcomprises at least 10, 15, 18, and preferably at least 20 nucleotides.Selectivity of hybridization occurs with a certain degree of specificityrather than being random. Appropriate stringency conditions whichpromote DNA hybridization are known to those skilled in the art, or canbe found in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6. For example, hybridization may occur at 30° C.in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS, preferably 37° C.in 500 mM NaCl, 500 mM trisodium citrate, 1% SDS, 35% formamide, and 100μg/ml denatured salmon sperm DNA (ssDNA), and more preferably 42° C. in250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

[0067] The stringency may be selected based on the conditions used inthe wash step. Wash step stringency conditions may be defined by saltconcentration and by temperature. Generally, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.By way of example, a stringent salt concentration for the wash step ispreferably less than about 30 mM NaCl and 3 mM trisodium citrate, andmore preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions will generally include temperatures ofa least about 25° C., more preferably at least about 68° C. In apreferred embodiment, the wash steps will be carried out at 42° C. in 15mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferredembodiment the wash steps are carried out at 68° C. in 15 mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. Variations on these conditions willbe readily apparent to those skilled in the art.

[0068] It will be appreciated that the invention includes nucleic acidmolecules encoding a Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-TRelated Polypeptide, including truncations of the polypeptides, allelicand species variants, and analogs of the polypeptides as describedherein. In particular, fragments of a nucleic acid of the invention arecontemplated that are a stretch of at least about 10, 15, or 18, andpreferably at least 20 nucleotides, more typically at least 50 to 200nucleotides but less than 2 kb. In an embodiment fragments are providedcomprising nucleic acid sequences encoding the cytosolic, transmembrane,and catalytic regions of Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide, (e.g. the sequences shown in SEQ.ID. NO.10, 11, or 12, respectively). It will further be appreciated thatvariant forms of the nucleic acid molecules of the invention which ariseby alternative splicing of an mRNA corresponding to a cDNA of theinvention are encompassed by the invention.

[0069] An isolated nucleic acid molecule of the invention whichcomprises DNA can be isolated by preparing a labeled nucleic acid probebased on all or part of the nucleic acid sequence shown in SEQ. ID. NO.1 or 3 (e.g.. bp 310-766), or 19 (for example SEQ ID NO: 15, 16, 17, 18,or 19). The labeled nucleic acid probe is used to screen an appropriateDNA library (e.g. a cDNA or genomic DNA library). For example, a cDNAlibrary can be used to isolate a cDNA encoding a Core 2b GlcNAc-TPolypeptide, or a Core 2b GlcNAc-T Related Polypeptide by screening thelibrary with the labeled probe using standard techniques. Alternatively,a genomic DNA library can be similarly screened to isolate a genomicclone encompassing a core2 gene. Nucleic acids isolated by screening ofa cDNA or genomic DNA library can be sequenced by standard techniques.

[0070] An isolated nucleic acid molecule of the invention that is DNAcan also be isolated by selectively amplifying a nucleic acid of theinvention. “Amplifying” or “amplification ” refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). Inparticular, it is possible to design synthetic oligonucleotide primersfrom the nucleotide sequence shown in SEQ. ID. NO. 1, 3, 10, 11, 12, or19 for use in PCR. A nucleic acid can be amplified from cDNA or genomicDNA using these oligonucleotide primers and standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis. cDNA maybe prepared from mRNA, by isolating total cellular mRNA by a variety oftechniques, for example, by using the guanidinium-thiocyanate extractionprocedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNAis then synthesized from the mRNA using reverse transcriptase (forexample, Moloney MLV reverse transcriptase available from Gibco/BRL,Bethesda, MD, or AMV reverse transcriptase available from SeikagakuAmerica, Inc., St. Petersburg, Fla).

[0071] An isolated nucleic acid molecule of the invention which is RNAcan be isolated by cloning a cDNA encoding a Core 2b GlcNAc-TPolypeptide, or a Core 2b GlcNAc-T Related Polypeptide into anappropriate vector which allows for transcription of the CDNA to producean RNA molecule which encodes a Core 2b GlcNAc-T Polypeptide, or a Core2b GlcNAc-T Related Polypeptide. For example, a cDNA can be cloneddownstream of a bacteriophage promoter, (e.g. a T7 promoter) in avector, cDNA can be transcribed in vitro with T7 polymerase, and theresultant RNA can be isolated by conventional techniques.

[0072] A nucleic acid molecule of the invention may be engineered usingmethods known in the art to alter the core-2b encoding sequence for avariety of purposes including modification of the cloning, processing,and/or expression of the gene product. Procedures such as DNA shufflingby random fragmentation and PCR reassembly of gene fragments andsynthetic oligonucleotides may be used to engineer the nucleic acidmolecules. Mutations may be introduced by oligonucleotide-mediatedsite-directed mutagenesis to create for example new restriction sites,alter glycosylation patterns, change codon preference, or produce splicevariants.

[0073] Nucleic acid molecules of the invention may be chemicallysynthesized using standard techniques. Methods of chemicallysynthesizing polydeoxynucleotides are known, including but not limitedto solid-phase synthesis which, like peptide synthesis, has been fullyautomated in commercially available DNA synthesizers (See e.g., Itakuraet al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No.4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

[0074] Determination of whether a particular nucleic acid molecule is acore2 or encodes a Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-TRelated Polypeptide can be accomplished by expressing the cDNA in anappropriate host cell by standard techniques, and testing the expressedpolypeptide in the methods described herein.

[0075] A core 2b GlcNAc-T CDNA or CDNA encoding a Core 2b GlcNAc-TPolypeptide, or a Core 2b GlcNAc-T Related Polypeptide can be sequencedby standard techniques, such as dideoxynucleotide chain termination orMaxam-Gilbert chemical sequencing, to determine the nucleic acidsequence and the predicted amino acid sequence of the encodedpolypeptide.

[0076] The nucleic acid molecules of the invention may be extended usinga partial nucleotide sequence and various PCR-based methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, restriction-site PCR which uses universal andnested primers to amplify unknown sequences from genomic DNA within acloning vector may be employed (See Sarkar, G, PCR Methods Applic.2:318-322, 1993). Inverse PCR which uses primers that extend indivergent directions to amplify unknown sequences from a circularizedtemplate may also be used. The template in inverse PCR is derived fromrestriction fragments adjacent to known sequences in human and yeastartificial chromosome DNA (See e.g. Lagerstrom, M., at al, PCR MethodsApplic. 1:111-119, 1991). Other methods for retrieving unknown sequencesare known in the art (e.g. Parker, J. D. et al, Nucleic Acids Res.19:305-306, 1991). In addition, PCR, nested primers, and PROMOTERFINDERlibraries (Clontech, Palo Alto, Calif.) may be used to walk genomic DNA.The method is useful in finding intron/exon junctions and avoids theneed to screen libraries.

[0077] It is preferable when screening for full-length cDNAs to uselibraries that have been size-selected to include larger cDNAs. Forsituations in which an oligo d(T) library does not yield a full-lengthcDNA, it is preferable to use random-primed libraries which ofteninclude sequences containing the 5′ regions of genes. Genomic librariesmay be useful for extending the sequence into 5′ non-translatedregulatory regions.

[0078] Commercially available capillary electrophoresis systems may beemployed to analyse the size or confirm the sequence of PCR orsequencing products. The system may use flowable polymers forelectrophoretic separation, four different nucleotide-specific,laser-stimulated fluorescent dyes, and a charge coupled device camerafor detection of the emitted wavelengths. Commercially availablesoftware (e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer) mayconvert the output/light intensity to electrical signal, and the entireprocess from loading of samples, and computer analysis and electronicdata display may be computer controlled. This procedure may beparticularly useful for sequencing small DNA fragments which may bepresent in limited amounts in a particular sample.

[0079] In accordance with one aspect of the invention, a nucleic acid isprovided comprising a core 2b GlcNAc-T regulatory sequence such as apromoter sequence. In particular, an isolated nucleic acid iscontemplated which comprises:

[0080] (i) a nucleic acid sequence having at least 60%, 75-80% identity,90%, and preferably at least 95% identity with the sequence of SEQ. ID.NO. 5 or 6;

[0081] (ii) nucleic acid sequences complementary to (i),

[0082] (iii) nucleic acid sequences differing from any of the nucleicacids of (i) or (ii) in codon sequences due to the degeneracy of thegenetic code;

[0083] (iv) a nucleic acid sequence comprising at least 10, 15, 18, andpreferably at least 20 nucleotides, and capable of hybridizing understringent conditions to a nucleic acid sequence of SEQ. ID. NO. 5 or 6,or to a degenerate form thereof,

[0084] (v) a fragment, or allelic or species variation of (i), (ii) or(iii).

[0085] In a preferred embodiment, the isolated nucleic acid comprises anucleic acid sequence of SEQ. ID. NO. 5 or 6, wherein T can also be U.

[0086] The invention contemplates nucleic acid molecules comprising allor a portion of a nucleic acid molecule of the invention comprising aregulatory sequence of acore 2b GlcNAc-T (e.g. SEQ ID Nos: 5 or 6)contained in appropriate vectors. The vectors may contain heterologousnucleic acid sequences. “Heterologous nucleic acid” refers to a nucleicacid not naturally located in the cell, or in a chromosomal site of thecell. Preferably, the heterologous nucleic acid includes a nucleic acidforeign to the cell.

[0087] In accordance with another aspect of the invention, the nucleicacid molecules isolated using the methods described herein are mutantcore2 gene alleles. For example, the mutant alleles may be isolated fromindividuals either known or proposed to have a genotype whichcontributes to the symptoms of a condition such as an inflammatorydisorder, cancer, or a gastrointestinal disorder. Mutant alleles andmutant allele products may be used in therapeutic and diagnostic methodsdescribed herein. For example, a cDNA of a mutant core 2b GlcNAc-T genemay be isolated using PCR as described herein, and the DNA sequence ofthe mutant allele may be compared to the normal allele to ascertain themutation(s) responsible for the loss or alteration of function of themutant gene product. A genomic library can also be constructed using DNAfrom an individual suspected of or known to carry a mutant allele, or acDNA library can be constructed using RNA from tissue known, orsuspected to express the mutant allele. A nucleic acid encoding a normalcore 2b GlcNAc-T gene or any suitable fragment thereof, may then belabeled and used as a probe to identify the corresponding mutant allelein such libraries. Clones containing mutant sequences can be purifiedand subjected to sequence analysis. In addition, an expression librarycan be constructed using cDNA from RNA isolated from a tissue of anindividual known or suspected to express a mutant core2 allele. Geneproducts from putatively mutant tissue may be expressed and screened,for example using antibodies specific for a Core 2b GlcNAc-TPolypeptide, or a Core 2b GlcNAc-T Related Polypeptide as describedherein. Library clones identified using the antibodies can be purifiedand subjected to sequence analysis.

[0088] Antisense molecules and ribozymes are contemplated within thescope of the invention. They may be prepared by any method known in theart for the synthesis of nucleic acid molecules. These includetechniques for chemically synthesizing oligonucleotides such as solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding core2b GlcNAc-T. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues. RNA molecules may be modified to increaseintracellular stability and half-life. Possible modifications include,but are not limited to, the addition of flanking sequences at the 5′and/or 3′ ends of the molecule or the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages within the backbone ofthe molecule. This concept is inherent in the production of PNAs and canbe extended in all of these molecules by the inclusion of nontraditionalbases such as inosine, queosine, and wybutosine, as well as acetyl-,methyl-, thio-, and similarly modified forms of adenine, cytidine,guanine, thymine, and uridine which are not as easily recognized byendogenous endonucleases.

[0089] Polypeptides of the Invention

[0090] The polypeptides of the invention are predominately expressed ingastrointestinal tissue (stomach, colon, intestine, testis), and areelevated in cancer (e.g. stomach and liver tumors).

[0091] The amino acid sequence of an isolated Core 2b GlcNAc-TPolypeptide of the invention comprises the sequence of SEQ.ID. NO. 2, 4,or 20. In addition to polypeptides comprising the amino acid sequence ofSEQ.ID. NO. 2, 4, or 20 the polypeptides of the present inventioninclude truncations, and analogs, allelic and species variations, andhomologs of Core 2b GlcNAc-T and truncations thereof as described herein(i.e Core 2b GlcNAc-T Related Polypeptide).

[0092] Truncated polypeptides may comprise peptides or fragments havingan amino acid sequence of at least five consecutive amino acids inSEQ.ID. NO. 2, 4, or 20 where no amino acid sequence of five or more,six or more, seven or more, or eight or more, consecutive amino acidspresent in the fragment is present in a polypeptide other than Core 2bGlcNAc-T. In an embodiment of the invention the fragment is a stretch ofamino acid residues of at least 12 to 50 contiguous amino acids,preferably 12 to 20 contiguous amino acids, from particular sequencessuch as the sequences shown in SEQ.ID. NO. 2, 4, or 20. The fragmentsmay be immunogenic and preferably are not immunoreactive with antibodiesthat are immunoreactive to polypeptides other than Core 2b GlcNAc-T. Thefragments may also have core 2b GicNAc-T activity. In an embodiment thefragments correspond to the cytosolic, transmembrane, or catalyticregions of a Core 2b GlcNAc-T Polypeptide, in particular the fragmentsshown in SEQ. ID. NO. 7, 8, or 9, respectively.

[0093] The truncated polypeptides may have an amino group (—NH2), ahydrophobic group (for example, carbobenzoxyl, dansyl, orT-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl(PMOC) group, or a macromolecule including but not limited tolipid-fatty acid conjugates, polyethylene glycol, or carbohydrates atthe amino terminal end. The truncated polypeptides may have a carboxylgroup, an amido group, a T-butyloxycarbonyl group, or a macromoleculeincluding but not limited to lipid-fatty acid conjugates, polyethyleneglycol, or carbohydrates at the carboxy terminal end.

[0094] The polypeptides of the invention may also include analogs ofCore 2b GlcNAc-T Polypeptide, and/or truncations thereof as describedherein, which may include, but are not limited to Core 2b GlcNAc-TPolypeptide, containing one or more amino acid substitutions,insertions, and/or deletions. Amino acid substitutions may be of aconserved or non-conserved nature. Conserved amino acid substitutionsinvolve replacing one or more amino acids of the Core 2b GlcNAc-T aminoacid sequence with amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade the resulting analog is preferably functionally equivalent to Core2b GlcNAc-T. Non-conserved substitutions involve replacing one or moreamino acids of the Core 2b GlcNAc-T amino acid sequence with one or moreamino acids that possess dissimilar charge, size, and/or hydrophobicitycharacteristics.

[0095] One or more amino acid insertions may be introduced into a Core2b GlcNAc-T Polypeptide. Amino acid insertions may consist of singleamino acid residues or sequential amino acids ranging from about 2 to 15amino acids in length.

[0096] Deletions may consist of the removal of one or more amino acids,or discrete portions from the amino acid sequence. The deleted aminoacids may or may not be contiguous. The lower limit length of theresulting analog with a deletion mutation is about 10 amino acids,preferably 100 amino acids.

[0097] An allelic variant of Core 2b GlcNAc-T at the polypeptide leveldiffers from one another by only one, or at most, a few amino acidsubstitutions. A species variation of a Core 2b GlcNAc-T Polypeptide isa variation which is naturally occurring among different species of anorganism.

[0098] The polypeptides of the invention include homologs of Core 2bPolypeptide and/or truncations thereof as described herein. Such Core 2bGlcNAc-T homologs include polypeptides whose amino acid sequences arecomprised of the amino acid sequences of Core 2b Polypeptide regionsfrom other species that hybridize under selective hybridizationconditions (see discussion of selective and in particular stringenthybridization conditions herein) with a probe used to obtain a Core 2bGlcNAc-T Polypeptide. These homologs will generally have the sameregions which are characteristic of a Core 2b GlcNAc-T Polypeptide. Itis anticipated that a polypeptide comprising an amino acid sequencewhich has at least 58% identity or at least 73% similarity, preferablyat least 60-65% identity or at least 80-85% similarity, more preferablyat least 70-80% identity or at least 90-95% similarity, most preferablyat least 95% identity or at least 99% similarity with the amino acidsequence of SEQ. ID. NO. 2, 4, 7, 8, 9, or 20 will be a homolog of aCore 2 Polypeptide. Polypeptides comprising an amino acid sequence whichhas at least 19% identify or 42% similarity, preferably at least 30-40%identity or at least 50% similarity, more preferably at least 50-60%identity or at least 60-70% similarity, and most preferably 70-80%identity or at least 80-95% similarity with the amino acid sequence ofSEQ. ID. NO. 2, 4, 7, 8, 9, or 20 are also anticipated to be homologs. Apercent amino acid sequence similarity or identity is calculated usingthe methods described herein, preferably the computer programs describedherein.

[0099] The invention also contemplates isoforms of the polypeptides ofthe invention. An isoform contains the same number and kinds of aminoacids as the polypeptide of the invention, but the isoform has adifferent molecular structure. The isoforms contemplated by the presentinvention preferably have the same properties as the polypeptide of theinvention as described herein.

[0100] The present invention also includes Core 2b GlcNAc-T Polypeptide,or a Core 2b GlcNAc-T Related Polypeptide conjugated with a selectedpolypeptide, or a marker polypeptide (see below), or otherglycosyltransferases to produce fusion polypeptides or chimericpolypeptides.

[0101] A Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T RelatedPolypeptide of the invention may be prepared using recombinant DNAmethods. Accordingly, the nucleic acids of the present invention havinga sequence which encodes a Core 2b GlcNAc-T Polypeptide, or a Core 2bRelated Polypeptide of the invention may be incorporated in a knownmanner into an appropriate vector which ensures good expression of thepolypeptide. Possible expression vectors include but are not limited tocosmids, plasmids, phages, or modified viruses (e.g. replicationdefective retroviruses, adenoviruses and adeno-associated viruses), solong as the vector is compatible with the host cell used.

[0102] The invention therefore contemplates a vector of the inventioncontaining a nucleic acid molecule of the invention, and the necessaryregulatory sequences for the transcription and translation of theinserted polypeptide-sequence. Suitable regulatory sequences may bederived from a variety of sources, including bacterial, fungal, viral,mammalian, or insect genes (For example, see the regulatory sequencesdescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Selection of appropriateregulatory sequences is dependent on the host cell chosen as discussedbelow, and may be readily accomplished by one of ordinary skill in theart. The necessary regulatory sequences may be supplied by the nativeCore 2b GlcNAc-T Polypeptide and/or its flanking regions.

[0103] The invention further provides a vector comprising a nucleic acidof the invention cloned into the expression vector in an antisenseorientation. That is, the DNA molecule is linked to a regulatorysequence in a manner which allows for expression, by transcription ofthe DNA molecule, of an RNA molecule which is antisense to the nucleicacid sequence of SEQ. ID. NO. 1, 3, 5, 6, 10, 11, 12,15, 16, 17, 18, or19. Regulatory sequences linked to the antisense nucleic acid can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance a viral promoterand/or enhancer, or regulatory sequences can be chosen which directtissue or cell type specific expression of antisense RNA.

[0104] The vectors of the invention may also contain a marker gene whichfacilitates the selection of host cells transformed or transfected witha recombinant molecule of the invention. Examples of marker genes aregenes encoding a polypeptide such as G418 and hygromycin which conferresistance to certain drugs, β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, or an immunoglobulin or portionthereof such as the Fc portion of an immunoglobulin preferably IgG. Themarkers can be introduced on a separate vector from the nucleic acid ofinterest.

[0105] The vectors may also contain genes that encode a fusion moietywhich provides increased expression of the recombinant polypeptide;increased solubility of the recombinant polypeptide; and aid in thepurification of the target recombinant polypeptide by acting as a ligandin affinity purification. For example, a proteolytic cleavage site maybe added to the target recombinant polypeptide to allow separation ofthe recombinant polypeptide from the fusion moiety subsequent topurification of the fusion polypeptide. Typical fusion expressionvectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E bindingpolypeptide, or polypeptide A, respectively, to the recombinantpolypeptide.

[0106] The vectors may be introduced into host cells to produce atransformed or transfected host cell. The terms “transfected” and“transfection” encompass the introduction of nucleic acid (e.g. avector) into a cell by one of many standard techniques. A cell is“transformed” by a nucleic acid when the transfected nucleic acideffects a phenotypic change. Prokaryotic cells can be transfected ortransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAF-dextran-mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

[0107] Suitable host cells include a wide variety of prokaryotic andeukaryotic host cells. For example, the polypeptides of the inventionmay be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus), yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, Gene Expression Technology: Methods,inEnzymology 185, Academic Press, San Diego, Calif. (1991).

[0108] A host cell may also be chosen which modulates the expression ofan inserted nucleic acid sequence, or modifies (e.g. glycosylation orphosphorylation) and processes (e.g. cleaves) the polypeptide in adesired fashion. Host systems or cell lines may be selected which havespecific and characteristic mechanisms for post-translational processingand modification of polypeptides. For example, eukaryotic host cellsincluding CHO, VERO, BHK, A431, HeLA, COS, MDCK, 293, 3T3, and W138 maybe used. For long-term high-yield stable expression of the polypeptide,cell lines and host systems which stably express the gene product may beengineered.

[0109] Host cells and in particular cell lines produced using themethods described herein may be particularly useful in screening andevaluating substances and compounds that modulate the activity of a Core2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T Related Polypeptide.

[0110] The polypeptides of the invention may also be expressed innon-human transgenic animals including but not limited to mice, rats,rabbits, guinea pigs, micro-pigs, goats, sheep, pigs, non-human primates(e.g. baboons, monkeys, and chimpanzees) (see Hammer et al. (Nature315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983),Brinster et al. (Proc Natl. Acad. Sci USA 82:44384442, 1985), Palmiterand Brinster (Cell. 41:343-345, 1985) and U.S. Pat. No. 4,736,866).Procedures known in the art may be used to introduce a nucleic acidmolecule of the invention encoding a Core 2b GlcNAc-T Polypeptide, or aCore 2b GlcNAc-T Related Polypeptide into animals to produce the founderlines of transgenic animals. Such procedures include pronuclearmicroinjection, retrovirus mediated gene transfer into germ lines, genetargeting in embryonic stem cells, electroporation of embryos, andsperm-mediated gene transfer.

[0111] The present invention contemplates a transgenic animal thatcarries the core 2b GlcNAc-T gene in all their cells, and animals whichcarry the transgene in some but not all their cells. The transgene maybe integrated as a single transgene or in concatamers. The transgene maybe selectively introduced into and activated in specific cell types (Seefor example, Lasko et al, 1992 Proc. Natl. Acad. Sci. USA 89: 6236). Thetransgene may be integrated into the chromosomal site of the endogenousgene by gene targeting. The transgene may be selectively introduced intoa particular cell type inactivating the endogenous gene in that celltype (See Gu et al Science 265: 103-106).

[0112] The expression of a recombinant Core 2b GlcNAc-T Polypeptide, ora Core 2b GlcNAc-T Related Polypeptide in a transgenic animal may beassayed using standard techniques. Initial screening may be conducted bySouthern Blot analysis, or PCR methods to analyze whether the transgenehas been integrated. The level of mRNA expression in the tissues oftransgenic animals may also be assessed using techniques includingNorthern blot analysis of tissue samples, in situ hybridization, andRT-PCR. Tissues may also be evaluated immunocytochemically usingantibodies against a Core 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-TRelated Polypeptide of the invention.

[0113] Polypeptides of the invention may also be prepared by chemicalsynthesis using techniques well known in the chemistry of polypeptidessuch as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc.85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987,Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,Stuttgart).

[0114] N-terminal or C-terminal fusion polypeptides or chimericpolypeptides comprising a Core 2b GlcNAc-T Polypeptide, or a Core 2bGlcNAc-T Related Polypeptide of the invention conjugated with othermolecules, such as polypeptides (e.g. markers or otherglycosyltransferases) may be prepared by fusing, through recombinanttechniques, the N-terminal or C-terminal of a Core 2b GlcNAc-TPolypeptide, or a Core 2b GlcNAc-T Related Polypeptide, and the sequenceof a selected polypeptide or marker polypeptide with a desiredbiological function. The resultant fusion polypeptides contain a Core 2bGlcNAc-T Polypeptide, or a Core 2b GlcNAc-T Related Polypeptide fused tothe selected polypeptide or marker polypeptide as described herein.Examples of polypeptides which may be used to prepare fusionpolypeptides include immunoglobulins, glutathione-S-transferase (GST),polypeptide A, hemagglutinin (HA), and truncated myc.

[0115] Antibodies

[0116] A polypeptide of the invention (including fragments) can be usedto prepare antibodies specific for the polypeptides. Antibodies can beprepared which bind a distinct epitope in an unconserved region of thepolypeptide. An unconserved region of the polypeptide is one that doesnot have substantial sequence homology to other polypeptides. A regionfrom a conserved region such as a well-characterized sequence can alsobe used to prepare an antibody to a conserved region of a polypeptide ofthe invention. Antibodies having specificity for a polypeptide of theinvention may also be raised from fusion polypeptides created byexpressing fusion polypeptides in host cells as described herein.

[0117] The invention can employ intact monoclonal or polyclonalantibodies, and immunologically active fragments (e.g. a Fab or (Fab)₂fragment), an antibody heavy chain, and antibody light chain, agenetically engineered single chain F_(v) molecule (Ladner et al, U.S.Pat. No. 4,946,778), humanized antibodies, or a chimeric antibody, forexample, an antibody which contains the binding specificity of a murineantibody, but in which the remaining portions are of human origin.Antibodies, including monoclonal and polyclonal antibodies, fragmentsand chimeras, may be prepared using methods known to those skilled inthe art.

[0118] Applications of the Nucleic Acid Molecules, Polypeptides, andAntibodies of the Invention

[0119] The nucleic acid molecules, Core 2b GlcNAc-T Polypeptide, or aCore 2b GlcNAc-T Related Polypeptide, and antibodies of the inventionmay be used in the prognostic and diagnostic evaluation of conditionsassociated with altered expression or activity of a polypeptide of theinvention or conditions requiring modulation of a nucleic acid orpolypeptide of the invention including inflammatory and proliferativedisorders, and gastrointestinal disorders, and the identification ofsubjects with a predisposition to such conditions (See below). Methodsfor detecting nucleic acid molecules and polypeptides of the invention,can be used to monitor such conditions (e.g. asthma, rheumatoidarthritis, inflammatory bowel disease, arteriosclerosis, septic shock,ARDS, cancer,) by detecting and localizing the polypeptides and nucleicacids. It would also be apparent to one skilled in the art that themethods described herein may be used to study the developmentalexpression of the polypeptides of the invention and, accordingly, willprovide further insight into the role of the polypeptides. Theapplications of the present invention also include methods for theidentification of substances or compounds that modulate the biologicalactivity of a polypeptide of the invention (See below). The substances,compounds, antibodies etc., may be used for the treatment of conditionsrequiring modulation of polypeptides of the invention. (See below).

[0120] Diagnostic Methods

[0121] A variety of methods can be employed for the diagnostic andprognostic evaluation of conditions requiring modulation of a nucleicacid or polypeptide of the invention (e.g. inflammatory disorders,gastrointestinal disorders, liver disorders, and cancer), and theidentification of subjects with a predisposition to such conditions.Such methods may, for example, utilize nucleic acid molecules of theinvention, and fragments thereof, and antibodies directed againstpolypeptides of the invention, including peptide fragments. Inparticular, the nucleic acids and antibodies may be used, for example,for: (1) the detection of the presence of core 2b GlcNAc-T mutations, orthe detection of either over- or under-expression of core 2b GlcNAc-TmRNA relative to a non-disorder state or the qualitative or quantitativedetection of alternatively spliced forms of core 2b GlcNAc-T transcriptswhich may correlate with certain conditions or susceptibility towardsuch conditions; or(2) the detection of either an over- or anunder-abundance of a polypeptide of the invention relative to anon-disorder state or the presence of a modified (e.g., less than fulllength) polypeptide of the invention which correlates with a disorderstate, or a progression toward a disorder state.

[0122] The methods described herein may be performed by utilizingpre-packaged diagnostic kits comprising for example, at least onespecific nucleic acid or antibody described herein, which may beconveniently used, e.g., in clinical settings, to screen and diagnosepatients and to screen and identify those individuals exhibiting apredisposition to developing a disorder.

[0123] Nucleic acid-based detection techniques and peptide detectiontechniques are described below. The samples that may. be analyzed usingthe methods of the invention include those that are known or suspectedto express core 2b -GlcNAc-T or contain a polypeptide of the invention.The methods may be performed on biological samples including but notlimited to cells, lysates of cells which have been incubated in cellculture, chromosomes isolated from a cell (e.g. a spread of metaphasechromosomes), genomic DNA (in solutions or bound to a solid support suchas for Southern analysis), RNA (in solution or bound to a solid supportsuch as for northern analysis), cDNA (in solution or bound to a solidsupport), and extract from cells or a tissue, and biological fluids suchas serum, urine, blood, and CSF. The samples may be derived from apatient or a culture.

[0124] Methods for Detecting Nucleic Acid Molecules of the Invention

[0125] The nucleic acid molecules of the invention allow those skilledin the art to construct nucleotide probes for use in the detection ofnucleic acid sequences of the invention in biological materials.Suitable probes include nucleic acid molecules based on nucleic acidsequences encoding at least 5 sequential amino acids from regions of theCore 2b GlcNAc-T Polypeptide, or a Core 2b GlcNAc-T Related Polypeptide(see SEQ. ID. No. 1, 3, 10, 11, 12, or 19), preferably they comprise 15to 50 nucleotides, more preferably 15 to 40 nucleotides, most preferably15-30 nucleotides. A nucleotide probe may be labeled with a detectablesubstance such as a radioactive label that provides for an adequatesignal and has sufficient half-life such as ³²P, ³H, ¹⁴C or the like.Other detectable substances that may be used include antigens that arerecognized by a specific labeled antibody, fluorescent compounds,enzymes, antibodies specific for a labeled antigen, and luminescentcompounds. An appropriate label may be selected having regard to therate of hybridization and binding of the probe to the nucleotide to bedetected and the amount of nucleotide available for hybridization.Labeled probes may be hybridized to nucleic acids on solid supports suchas nitrocellulose filters or nylon membranes as generally described inSambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).The nucleic acid probes may be used to detect core 2b GlcNAc-T genes,preferably in human cells. The nucleotide probes may also be useful forexample in the diagnosis or prognosis of conditions such as inflammatorydisorders, gastrointestinal disorders, liver disorders, kidneydisorders, and cancer, and in monitoring the progression of theseconditions, or monitoring a therapeutic treatment.

[0126] The probe may be used in hybridization techniques to detect acore 2b GlcNAc-T gene. The technique generally involves contacting andincubating nucleic acids (e.g. recombinant DNA molecules, cloned genes)obtained from a sample from a patient or other cellular source with aprobe of the present invention under conditions favourable for thespecific annealing of the probes to complementary sequences in thenucleic acids. After incubation, the non-annealed nucleic acids areremoved, and the presence of nucleic acids that have hybridized to theprobe if any are detected.

[0127] The detection of nucleic acid molecules of the invention mayinvolve the amplification of specific gene sequences using anamplification method (e.g. PCR), followed by the analysis of theamplified molecules using techniques known to those skilled in the art.Suitable primers can be routinely designed by one of skill in the art.For example, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer Analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

[0128] Genomic DNA may be used in hybridization or amplification assaysof biological samples to detect abnormalities involving core 2b GlcNAc-Tstructure, including point mutations, insertions, deletions, andchromosomal rearrangements. For example, direct sequencing, singlestranded conformational polymorphism analyses, heteroduplex analysis,denaturing gradient gel electrophoresis, chemical mismatch cleavage, andoligonucleotide hybridization may be utilized.

[0129] Genotyping techniques known to one skilled in the art can be usedto type polymorphisms that are in close proximity to the mutations in acore 2b GlcNAc-T gene. The polymorphisms may be used to identifyindividuals in families that are likely to carry mutations. If apolymorphism exhibits linkage disequalibrium with mutations in the core2b GlcNAc-T gene, it can also be used to screen for individuals in thegeneral population likely to carry mutations. Polymorphisms which may beused include restriction fragment length polymorphisms (RFLPs),single-base polymorphisms, and simple sequence repeat polymorphisms(SSLPs).

[0130] A probe or primer of the invention may be used to directlyidentify RFLPs. A probe or primer of the invention can additionally beused to isolate genomic clones such as YACs, BACs, PACs, cosmids, phageor plasmids. The DNA in the clones can be screened for SSLPs usinghybridization or sequencing procedures.

[0131] Hybridization and amplification techniques described herein maybe used to assay qualitative and quantitative aspects of core 2bGlcNAc-T expression. For example, RNA may be isolated from a cell typeor tissue known to express core 2b GlcNAc-T and tested utilizing thehybridization (e.g. standard Northern analyses) or PCR techniquesreferred to herein. The techniques may be used to detect differences intranscript size that may be due to normal or abnormal alternativesplicing. The techniques may be used to detect quantitative differencesbetween levels of full length and/or alternatively splice transcriptsdetected in normal individuals relative to those individuals exhibitingsymptoms of a disease.

[0132] The primers and probes may be used in the above described methodsin situ i.e directly on tissue sections (fixed and/or frozen) of patienttissue obtained from biopsies or resections.

[0133] Oligonucleotides or longer fragments derived from any of thenucleic acid molecules of the invention may be used as targets in amicroarray. The microarray can be used to simultaneously monitor theexpression levels of large numbers of genes and to identify geneticvariants, mutations, and polymorphisms. The information from themicroarray may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, to determinepredisposition to certain conditions, to treat the disorder, and todevelop and monitor the activities of therapeutic agents.

[0134] The preparation, use, and analysis of microarrays are well knownto a person skilled in the art. (See, for example, Brennan, T. M. et al.(1995) U.S. Pat. No. 5,474,796; Schena, et al. (1996) Proc. Natl. Acad.Sci. 93:10614-10619; Baldeschweiler et al. (1995), PCT ApplicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0135] Methods for Detecting Polypeptides

[0136] Antibodies specifically reactive with a Core 2b GlcNAc-TPolypeptide, a Core 2b GlcNAc-T Related Polypeptide, or derivatives,such as enzyme conjugates or labeled derivatives, may be used to detectCore 2b GlcNAc-T Polypeptides or Core 2b GlcNAc-T Related Polypeptidesin various biological materials. They may be used as diagnostic orprognostic reagents and they may be used to detect abnormalities in thelevel of Core 2b GlcNAc-T Polypeptides or Core 2b GlcNAc-T RelatedPolypeptides, expression, or abnormalities in the structure, and/ortemporal, tissue, cellular, or subcellular location of the polypeptides.Antibodies may also be used to screen potentially therapeutic compoundsin vitro to determine their effects on a condition such as aninflammatory disorder, cancer, or gastrointestinal disorders. In vitroimmunoassays may also be used to assess or monitor the efficacy ofparticular therapies. The antibodies of the invention may also be usedin vitro to determine the level of Core 2b GlcNAc-T Polypeptide or Core2b GlcNAc-T Related Polypeptide expression in cells geneticallyengineered to produce a Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-TRelated Polypeptide.

[0137] The antibodies may be used in any known immunoassays that rely onthe binding interaction between an antigenic determinant of apolypeptide of the invention, and the antibodies. Examples of suchassays are radioimmunoassays, enzyme immunoassays (e.g. ELISA),immunofluorescence, immunoprecipitation, latex agglutination,hemagglutination, and histochemical tests. The antibodies may be used todetect and quantity polypeptides of the invention in a sample in orderto determine their role in particular cellular events or pathologicalstates, and to diagnose and treat such pathological states.

[0138] In particular, the antibodies of the invention may be used inimmuno-histochemical analyses, for example, at the cellular andsub-subcellular level, to detect a polypeptide of the invention, tolocalise it to particular cells and tissues, and to specific subcellularlocations, and to quantitate the level of expression.

[0139] Cytochemical techniques known in the art for localizing antigensusing light and electron microscopy may be used to detect a polypeptideof the invention. Generally, an antibody of the invention may be labeledwith a detectable substance and a polypeptide may be localised intissues and cells based upon the presence of the detectable substance.Various methods of labeling polypeptides are known in the art and may beused. Examples of detectable substances include, but are not limited to,the following: radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I), ¹³¹I,fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),luminescent labels such as luminol; enzymatic labels (e.g., horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase,acetylcholinesterase), biotinyl groups (which can be detected by markedavidin e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or calorimetric methods),predetermined polypeptide epitopes recognized by a.secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). In some embodiments,labels are attached via spacer arms of various lengths to reducepotential steric hindrance. Antibodies may also be coupled to electrondense substances, such as ferritin or colloidal gold, which are readilyvisualised by electron microscopy.

[0140] The antibody or sample may be immobilized on a carrier or solidsupport which is capable of immobilizing cells, antibodies, etc. Forexample, the carrier or support may be nitrocellulose, or glass,polyacrylamides, gabbros, and magnetite. The support material may haveany possible configuration including spherical (e.g. bead), cylindrical(e.g. inside surface of a test tube or well, or the external surface ofa rod), or flat (e.g. sheet, test strip). Indirect methods may also beemployed in which the primary antigen-antibody reaction is amplified bythe introduction of a second antibody, having specificity for theantibody reactive against a polypeptide of the invention. By way ofexample, if the antibody having specificity against a polypeptide of theinvention is a rabbit IgG antibody, the second antibody may be goatanti-rabbit gamma-globulin labeled with a detectable substance asdescribed herein.

[0141] Where a radioactive label is used as a detectable substance, apolypeptide of the invention may be localized by radioautography. Theresults of radioautography may be quantitated by determining the densityof particles in the radioautographs by various optical methods, or bycounting the grains.

[0142] A polypeptide of the invention may also be detected by assayingfor Core 2b GlcNAc-T activity as described herein. For example, a samplemay be reacted with an acceptor substrate and a sugar donor underconditions where a Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-TRelated Polypeptide is capable of transferring the sugar donor to theacceptor substrate to produce a sugar donor-acceptor substrate complex.

[0143] Methods for Identifying or Evaluating Substances/Compounds

[0144] The methods described herein are designed to identiify substancesand compounds that modulate the expression or biological activity of aCore 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-T Related Polypeptideincluding substances that interfere with, or enhance the expression oractivity of a Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-T RelatedPolypeptide.

[0145] Substances and compounds identified using the methods of theinvention include but are not limited to peptides such as solublepeptides including Ig-tailed fusion peptides, members of random peptidelibraries and combinatorial chemistry-derived molecular libraries madeof D- and/or L-configuration amino acids, phosphopeptides (includingmembers of random or partially degenerate, directed phosphopeptidelibraries), antibodies [e.g. polyclonal, monoclonal, humanized,anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab,F(ab)₂, and Fab expression library fragments, and epitope-bindingfragments thereof)], polypeptides, nucleic acids, carbohydrates, amonosaccharide, an oligosaccharide or polysaccharide, and small organicor inorganic molecules. A substance or compound may be an endogenousphysiological compound or it may be a natural or synthetic compound.

[0146] Substances which modulate a Core 2b GlcNAc-T Polypeptide or Core2b GlcNAc-T Related Polypeptide can be identified based on their abilityto associate with a Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-TRelated Polypeptide. Therefore, the invention also provides methods foridentifying substances that associate with a Core 2b GlcNAc-TPolypeptide or Core 2b GlcNAc-T Related Polypeptide. Substancesidentified using the methods of the invention may be isolated, clonedand sequenced using conventional techniques. A substance that associateswith a polypeptide of the invention may be an agonist or antagonist ofthe biological or immunological activity of a polypeptide of theinvention.

[0147] The term “agonist” refers to a molecule that increases the amountof, or prolongs the duration of, the activity of the polypeptide. Theterm “antagonist” refers to a molecule which decreases the biological orimmunological activity of the polypeptide. Agonists and antagonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculesthat associate with a polypeptide of the invention.

[0148] Substances which can associate with a Core 2b GlcNAc-TPolypeptide or Core 2b GlcNAc-T Related Polypeptide may be identified byreacting a Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-T RelatedPolypeptide with a test substance which potentially associates with aCore 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-T Related Polypeptide,under conditions which permit the association, and removing and/ordetecting the associated Core 2b GlcNAc-T Polypeptide or Core 2bGlcNAc-T Related Polypeptide and substance. Substance-polypeptidecomplexes, free substance, or non-complexed polypeptides may be assayed.Conditions which permit the formation of substance-polypeptide complexesmay be selected having regard to factors such as the nature and amountsof the substance and the polypeptide.

[0149] The substance-polypeptide complex, free substance ornon-complexed polypeptides may be isolated by conventional isolationtechniques, for example, salting out, chromatography, electrophoresis,gel filtration, fractionation, absorption, polyacrylamide gelelectrophoresis, agglutination, or combinations thereof. To facilitatethe assay of the components, antibody against a polypeptide of theinvention or the substance, or labeled polypeptide, or a labeledsubstance may be utilized. The antibodies, polypeptides, or substancesmay be labeled with a detectable substance as described above.

[0150] A Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-T RelatedPolypeptide, or the substance used in the method of the. invention maybe insolubilized. For example, a polypeptide, or substance may be boundto a suitable carrier such as agarose, cellulose, dextran, Sephadex,Sepharose, carboxymethyl cellulose polystyrene, filter paper,ion-exchange resin, plastic film, plastic tube, glass beads,polyamine-methyl vinyl-ether-maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carriermay be in the shape of, for example, a tube, test plate, beads, disc,sphere etc. The insolubilized polypeptide or substance may be preparedby reacting the material with a suitable insoluble carrier using knownchemical or physical methods, for example, cyanogen bromide coupling.

[0151] The invention also contemplates a method for evaluating acompound for its ability to modulate the biological activity of apolypeptide of the invention, by assaying for an agonist or antagonist(i.e. enhancer or inhibitor) of the association of the polypeptide witha substance which associates with the polypeptide. The basic method forevaluating if a compound is an agonist or antagonist of the associationof a polypeptide of the invention and a substance that associates withthe polypeptide, is to prepare a reaction mixture containing thepolypeptide and the substance under conditions which permit theformation of substance-polypeptide complexes, in the presence of a testcompound. The test compound may be initially added to the mixture, ormay be added subsequent to the addition of the polypeptide andsubstance. Control reaction mixtures without the test compound or with aplacebo are also prepared. The formation of complexes is detected andthe formation of complexes in the control reaction but not in thereaction mixture indicates that the test compound interferes with theinteraction of the polypeptide and substance. The reactions may becarried out in the liquid phase or the polypeptide, substance, or testcompound may be immobilized as described herein.

[0152] It will be understood that the agonists and antagonists i.e.inhibitors and enhancers, that can be assayed using the methods of theinvention may act on one or more of the interaction sites on thepolypeptide or substance including agonist binding sites, competitiveantagonist binding sites, non-competitive antagonist binding sites orallosteric sites.

[0153] The invention also makes it possible to screen for antagoniststhat inhibit the effects of an agonist of the interaction of apolypeptide of the invention with a substance which is capable ofassociating with the polypeptide. Thus, the invention may be used toassay for a compound that competes for the same interacting site of apolypeptide of the invention.

[0154] Substances that modulate a Core 2b GlcNAc-T Polypeptide or Core2b GlcNAc-T Related Polypeptide of the invention can be identified basedon their ability to interfere with or enhance the activity of a Core 2bGlcNAc-T Polypeptide or Core 2b GlcNAc-T Related Polypeptide. Therefore,the invention provides a method for evaluating a compound for itsability to modulate the activity of a Core 2b GlcNAc-T Polypeptide orCore 2b GlcNAc-T Related Polypeptide comprising (a) reacting an acceptorand a sugar donor for a Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-TRelated Polypeptide in the presence of a test substance; (b) measuringthe amount of sugar donor transferred to acceptor, and (c) carrying outsteps (a) and (b) in the absence of the test substance to determine ifthe substance interferes with or enhances transfer of the sugar donor tothe acceptor. by the Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-TRelated Polypeptide.

[0155] Suitable acceptors for use in the methods of the invention are asaccharide, oligosaccharides, polysaccharides, glycopeptides,glycopolypeptides, or glycolipids which are either synthetic withlinkers at the reducing end or naturally occurring structures, forexample, asialo-agalacto-fetuin glycopeptide. Acceptors will generallyinclude β-D-galactosyl-1,3-N-acetyl-D-galactosaminyl-.

[0156] The sugar donor may be a nucleotide sugar,dolichol-phosphate-sugar or dolichol-pyrophosphate-oligosaccharide, forexample, uridine diphospho-N-acetylglucosamine (UDP-GlcNAc), orderivatives or analogs thereof. The Core 2b GlcNAc-T Polypeptide or Core2b GlcNAc-T Related Polypeptide may be obtained from natural sources orproduced used recombinant methods as described herein.

[0157] The acceptor or sugar donor may be labeled with a detectablesubstance as. described herein, and the interaction of the polypeptideof the invention with the acceptor and sugar donor will give rise to adetectable change. The detectable change may be calorimetric,photometric, radiometric, potentiometric, etc. The activity of a Core 2bGlcNAc-T Polypeptide or Core 2b GlcNAc-T Related Polypeptide of theinvention may also be determined using methods based on HPLC (Koendermanet al., FEBS Lett. 222:42, 1987) or methods employed syntheticoligosaccharide acceptors attached to hydrophobic aglycones (Palcic etal Glycoconjugate 5:49, 1988; and Pierce et al, Biochem. Biophys. Res.Comm. 146: 679, 1987).

[0158] The Core 2b GlcNAc-T Polypeptide or Core 2b GlcNAc-T RelatedPolypeptide is reacted with the acceptor and sugar donor at a pH andtemperature and in the presence of a metal cofactor, usually a divalentcation like manganese, effective for the polypeptide to transfer thesugar donor to the acceptor, and where one of the components is labeled,to produce a detectable change. It is preferred to use a buffer with theacceptor and sugar donor to maintain the pH within the pH rangeeffective for the polypeptides. The buffer, acceptor, and sugar donormay be used as an assay composition. Other compounds such as EDTA anddetergents may be added to the assay composition.

[0159] The reagents suitable for applying the methods of the inventionto evaluate compounds that modulate a Core 2b GlcNAc-T Polypeptide orCore 2b GlcNAc-T Related Polypeptide may be packaged into convenientkits providing the necessary materials packaged into suitablecontainers. The kits may also include suitable supports useful inperforming the methods of the invention.

[0160] Substances that modulate a Core 2b GlcNAc-T Polypeptide or Core2b GlcNAc-T Related Polypeptide can also be identified by treatingimmortalized cells which express the polypeptide with a test substance,and comparing the morphology of the cells with the morphology of thecells in the absence of the substance and/or with immortalized cellswhich do not express the polypeptide. Examples of immortalized cellsthat can be used include lung epithelial cell lines such as MvlLu orHEK293 (human embryonic kidney) transfected with a vector containing anucleic acid of the invention. In the absence of an inhibitor the cellsshow signs of morphologic transformation (e.g. fibroblastic morphology,spindle shape and pile up; the cells are less adhesive to substratum;there is less cell to cell contact in monolayer culture; there isreduced growth-factor requirements for survival and proliferation; thecells grow in soft-agar of other semi-solid medium; there is a lack ofcontact inhibition and increased apoptosis in low-serum high densitycultures; there is enhanced cell motility, and there is invasion intoextracellular matrix and secretion of proteases). Substances thatinhibit one or more phenotypes may be considered an inhibitor.

[0161] A substance that inhibits a Core 2b GlcNAc-T Polypeptide or Core2b GlcNAc-T Related Polypeptide may be identified by treating a cellwhich expresses the polypeptide with a test substance, and assaying forcomplex core 2-based Glinked structures (e.g. repeating Gal[β]1-4GlcNAc[β]) associated with the cell. The complex core 2-basedO-linked structures can be assayed using a substance that binds to thestructures (e.g. antibodies). Cells that have not been treated with thesubstance or which do not express the polypeptide may be employed ascontrols.

[0162] Substances which inhibit transcription or translation of a core2b gene may be identified by transfecting a cell with an expressionvector comprising a recombinant molecule of the invention, including areporter gene, in the presence of a test substance and comparing thelevel of expression of the Core 2b GlcNAc-T Polypeptide or Core 2bGlcNAc-T Related Polypeptide, or the expression of the protein encodedby the reporter gene, with a control cell transfected with the nucleicacid molecule in the absence of the substance. The method can be used toidentify transciption and translation inhibitors of a core 2b gene.

[0163] Compositions and Treatments

[0164] The substances or compounds identified by the methods describedherein, polypeptides, nucleic acid molecules, and antibodies of theinvention may be used for modulating the biological activity of a Core2bGlcNAc-T polypeptide or a Core2b GlcNAc-T Related Polypeptide, and theymay be used in the treatment of conditions mediated by Core 2bGlcNAc-Transferases. In particular, they may be used to modulatecellular adhesion associated with a number of disorders includinginflammatory disorders, and cancer.

[0165] The terms “inflammatory” refers to reactions of both the specificand non-specific defense systems. A specific defense system reaction isa specific immune system reaction to an antigen. Examples of thesereactions include antibody response to antigens such as viruses, anddelayed-type hypersensitivity. A non-specific defense system reaction isan inflammatory response mediated by leukocytes (including macrophages,eosinophils, and neutrophils) generally incapable of immunologicalmemory. Examples of non-specific reactions include the immediateswelling after a bee sting, and the collection of peripheral mononuclearleukocytes at sites of bacterial infection (pulmonary infiltrates inbacterial pneumonia and pus formation is abscesses).

[0166] Treatable disorders include rheumatoid arthritis, post-ischemicleukocyte-mediated tissue damage (reperfusion injury), frost-bite injuryor shock, acute leukocyte-mediated lung injury (e.g. adult respiratorydistress syndrome (ARDS)), asthma, traumatic shock, septic shock,nephritis, and acute and chronic inflammation including atopicdermatitis, psoriasis, and inflammatory bowel disease. Variouspiatelet-mediated pathologies such as atherosclerosis and clotting canalso be treated. The substances and compounds may be useful inminimizing tissue damage accompanying thrombotic disorders. For example,the substances, compounds, antibodies etc. can be of therapeutic valuein individuals who have recently experienced stroke, myocardialinfarctions, deep vein thrombosis, pulmonary embolism, etc. or inpre-thrombolytic therapy. Inhibitors of Core 2b GlcNAc-T may be usefulin reducing angiogenesis as well as leukocyte adhesion and entry intoinflamed tissue.

[0167] A substance, compound, etc. may be used to treat the secondaryeffects (e.g. pathological tissue destruction, and/or widespreadmicrocirculatory thrombi and diffuse inflammation) of septic shock ordisseminated intravascular coagulation (DIC). Substances compounds, etc.herein may inhibit leukocyte emigration and mitigate tissue damage.

[0168] A substance, compound, etc. may also be useful in treatingtraumatic shock and associated acute tissue injury. Inhibitorysubstances, compounds etc. may be administered locally or systemicallyto control tissue damage associated with injuries.

[0169] The substances or compounds identified by the methods describedherein, antibodies, and polypeptides, and nucleic acid molecules oftheinvention may be useful in the prevention and treatment of tumors. Tumormetastasis may be inhibited or prevented by inhibiting the adhesion ofcirculating cancer cells. The substances, compounds, etc. of theinvention may be especially useful in the treatment of various forms ofneoplasia such as leukemias, lymphomas, melanomas, adenomas, sarcomas,and carcinomas of solid tissues in patients. In particular thecomposition may be used for treating malignant melanoma, pancreaticcancer, cervico-uterine cancer, cancer of the liver, kidney, stomach,lung, rectum, breast, bowel, gastric, thyroid, neck, cervix, salivarygland, bile duct, pelvis, mediastinum, urethra, bronchogenic, bladder,esophagus and colon, and Kaposi's Sarcoma which is a form of cancerasociated with HIV-infected patients with Acquired Immune DeficiencySyndrome (AIDS). The substances etc. are particularly useful in theprevention and treatment of tumors of the gastrointestinal tractincluding adenomatous polyps (e.g. familial polyposis and Gardener'ssyndrome), cancer of the kidney, colon, and small intestine, tumors ofthe liver, and the metastases derived from these tumors.

[0170] Gastrointestinal disorders that may be prevented or treated usingthe substances or compounds identified by the methods described herein,antibodies, and polypeptides, and nucleic acid molecules of theinvention include ascites, cholelithiasis, cholecystitis, cirrhosis,Crohn's disease, diverticulitis, fulminant hepatitis, gastritis, gastricand duodenal ulcers, hepatorenal syndrome, irritable bowel syndrome,jaundice, pancreatitis, and ulcerative colitis.

[0171] Other conditions that are treatable with a substance or compoundidentified in accordance with the methods described herein, antibodies,polypeptides, or nucleic add molecules of the invention areproliferative disorders (e.g. microbial or parasitic infections), kidneydisorders, diabetes, and cardiomyopathy. They may also be used tomodulate T-cell activation and immunodeficienty due to theWiskott-Aldrich syndrome or AIDS, or to stimulate hematopoieticprogenitor cell growth, and/or confer protection against chemotherapyand radiation therapy in a subject.

[0172] Accordingly, the substances, antibodies, and compounds may beformulated into pharmaceutical compositions for administration tosubjects in a biologically compatible form suitable for administrationin vivo. By “biologically compatible form suitable for administration invivo” is meant a form of the substance to be administered in which anytoxic effects are outweighed by the therapeutic effects. The substancesmay be administered to living organisms including humans, and animals.Administration of a therapeutically active amount of the pharmaceuticalcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. For example, a therapeutically active amount of a substance mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of antibody to elicit adesired response in the individual. Dosage regima may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

[0173] The active substance may be administered in a convenient mannersuch as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, the activesubstance may be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions that mayinactivate the compound.

[0174] The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe substances or compounds in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

[0175] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of an inhibitor of a polypeptideof the invention, such labeling would include amount, frequency, andmethod of administration.

[0176] The nucleic acid molecules encoding Core2b GlcNAc-T Polypeptidesor any fragment thereof, or antisense sequences may be used fortherapeutic purposes. Antisense to a nucleic acid molecule encoding apolypeptide of the invention may be used in situations to block thesynthesis of the polypeptide. In particular, cells may be transformedwith sequences complementary to nucleic acid molecules encoding Core 2bGlcNAc-T Polypeptide. Thus, antisense sequences may be used to modulateCore 2b GlcNAc-T activity or to achieve regulation of gene function.Sense or antisense oligomers or larger fragments, can be designed fromvarious locations along the coding or regulatory regions of sequencesencoding a polypeptide of the invention.

[0177] Expression vectors may be derived from retroviruses,adenoviruses, herpes or vaccinia viruses or from various bacterialplasmids for delivery of nucleic acid sequences to the target organ,tissue, or cells. Vectors that express antisense nucleic acid sequencesof core 2b GlcNAc-T can be constructed using techniques well known tothose skilled in the art (see for example, Sambrook et al. (supra)).

[0178] Genes encoding core2b GlcNAc-T can be turned off by transforminga cell or tissue with expression vectors that express high levels of anucleic acid molecule or fragment thereof which encodes a polypeptide ofthe invention. Such constructs may be used to introduce untranslatablesense or antisense sequences into a cell. Even if they do not integrateinto the DNA, the vectors may continue to transcribe RNA molecules untilall copies are disabled by endogenous nucleases. Transient expressionmay last for extended periods of time (e.g a month or more) with anon-replicating vector or if appropriate replication elements are partof the vector system.

[0179] Modification of gene expression may be achieved by designingantisense molecules, DNA, RNA, or PNA, to the control regions of a core2b GlcNAc-T gene i.e. the promoters, enhancers, and introns. Preferablythe antisense molecules are oligonucleotides derived from thetranscription initiation site (e.g. between positions—10 and +10 fromthe start site). Inhibition can also be achieved by using triple-helixbase-pairing techniques. Triple helix pairing causes inhibition of theability of the double helix to open sufficiently for the binding ofpolymerases, transcription factors, or regulatory molecules (see Gee J.E. et al (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Anantisense molecule may also be designed to block translation of mWNA byinhibiting binding of the transcript to the ribosomes.

[0180] Ribozymes, enzymatic RNA molecules, may be used to catalyze thespecific cleavage of RNA. Ribozyme action involves sequence-specifichybridization of the ribozyme molecule to complementary target RNA,followed by endonucleolytic cleavage. For example, hammerhead motifribozyme molecules may be engineered that can specifically andefficiently catalyze endonucleolytic cleavage of sequences encoding apolypeptide of the invention.

[0181] Specific ribosome cleavage sites within any RNA target may beinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Short RNA sequences of between 15 and 20 ribonucleotides correspondingto the region of the cleavage site of the target gene may be evaluatedfor secondary structural features which may render the oligonucleotideinoperable. The suitability of candidate targets may be evaluated bytesting accessibility to hybridization with complementaryoligonucleotides using ribonuclease protection assays.

[0182] Methods for introducing vectors into cells or tissues includethose methods discussed herein and which are suitable for in vivo, invitro and ex vivo therapy. For ex vivo therapy, vectors may beintroduced into stem cells obtained from a patient and clonallypropagated for autologous transplant into the same patient (See U.S.Pat. Nos. 5,399,493 and 5,437,994). Delivery by transfection and byliposome are well known in the art.

[0183] The nucleic acid molecules disclosed herein may also be used inmolecular biology techniques that have not yet been developed, providedthe new techniques rely on properties of nucleotide sequences that arecurrently known, including but not limited to such properties as thetriplet genetic code and specific base pair interactions.

[0184] The invention also provides methods for studying the function ofa Core2b GlcNAc-T polypeptide or a Core2b GlcNAc-T Related Polypeptide.Cells, tissues, and non-human animals lacking in core2b GlcNAc-Texpression or partially lacking in core2b GlcNAc-T expression may bedeveloped using recombinant expression vectors of the invention havingspecific deletion or insertion mutations in thecore2b GlcNAc-T gene. Arecombinant expression vector may be used to inactivate or alter theendogenous gene by homologous recombination, and thereby create a core2GlcNAc-T deficient cell, tissue, or animal.

[0185] Null alleles may be generated in cells, such as embryonic stemcells by deletion mutation. A recombinant core2b GlcNAc-T gene may alsobe engineered to contain an insertion mutation which inactivates core2bGlcNAc-T Such a construct may then be introduced into a cell, such as anembryonic stem cell, by a technique such as transfection,electroporation, injection etc. Cells lacking an intact core2b GlcNAc-Tgene may then be identified, for example by Southern blotting, NorthernBlotting or by assaying for expression of a polypeptide of the inventionusing the methods described herein. Such cells may then be used togenerate transgenic non-human animals deficient in core2b GlcNAc-T.Germline transmission of the mutation may be achieved, for example, byaggregating the embryonic stem cells with early stage embryos, such as 8cell embryos, in vitro; transferring the resulting blastocysts intorecipient females; and, generating gerniline transmission of theresulting aggregation chimeras. Such a mutant animal may be used todefine specific cell populations, developmental patterns and in vivoprocesses, normally dependent on core2b GlcNAc-T expression.

[0186] The invention thus provides a transgenic non-human mammal all ofwhose germ cells and somatic cells contain a recombinant expressionvector that inactivates or alters a gene encoding a Core2b GlcNAc-Tpolypeptide or a Core2b GlcNAc-T Related Polypeptide. In an embodimentthe invention provides a transgenic nonhuman mammal all of whose germcells and somatic cells contain a recombinant expression vector thatinactivates or alters a gene encoding a Core2b GlcNAc-T polypeptide or aCore2b GlcNAc-T Related Polypeptide resulting in a Core2b GlcNAc-Tpolypeptide or a Core2b GlcNAc-T Related Polypeptide associatedpathology. Further the invention provides a transgenic non-human mammalwhich does not express or has altered expression of a Core2b GlcNAc-Tpolypeptide or a Core2b GlcNAc-T Related Polypeptide of the invention.In an embodiment, the invention provides a transgenic non-human mammalwhich does not express or has altered expression of a Core2b GlcNAc-Tpolypeptide or a Core2b GlcNAc-T Related Polypeptide of the inventionresulting in a Core2b GlcNAc-T polypeptide or a Core2b GlcNAc-T RelatedPolypeptide associated pathology. A Core2b GlcNAc-T polypeptide or aCore2b GlcNAc-T Related Polypeptide pathology refers to a phenotypeobserved for a Core2b GlcNAc-T polypeptide or a Core2b GlcNAc-T RelatedPolypeptide homozygous or heterozygous mutant.

[0187] A transgenic non-human animal includes but is not limited tomouse, rat, rabbit, sheep, hamster, dog, cat, goat, and monkey,preferably mouse.

[0188] The invention also provides a transgenic non-human animal assaysystem which provides a model system for testing for an agent thatreduces or inhibits a pathology associated with a Core2b GlcNAc-Tpolypeptide or a Core2b GlcNAc-T Related Polypeptide, preferably aCore2b GlcNAc-T polypeptide or a Core2b GlcNAc-T Related Polypeptideassociated pathology, comprising:

[0189] (a) administering the agent to a transgenic non-human animal ofthe invention; and

[0190] (b) determining whether said agent reduces or inhibits thepathology (e.g. Core2b GlcNAc-T polypeptide or a Core2b GlcNAc-T RelatedPolypeptide associated pathology) in the transgenic non-human animalrelative to a transgenic non-human animal of step (a) which has not beenadministered the agent.

[0191] The agent may be useful in the treatment and prophylaxis ofconditions such as inflammatory disorders or cancer as discussed herein.The agents may also be incorporated in a pharmaceutical composition asdescribed herein.

[0192] Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or with experimental animals,such as by calculating the ED₅₀ (the dose therapeutically effective in50% of the population) or LD₅₀ (the dose lethal to 50% of thepopulation) statistics. The therapeutic index is the dose ratio oftherapeutic to toxic effects and it can be expressed as the ED₅₀/LD₅₀ratio. Pharmaceutical compositions which exhibit large therapeuticindices are preferred.

[0193] A polypeptide of the invention may be used to support thesurvival, growth, migration, and/or differentiation of cells expressingthe polypeptide. Thus, a polypeptide of the invention may be used as asupplement to support, for example cells in culture.

[0194] Methods for Preparing Oligosaccharides

[0195] The invention relates to a method for preparing anoligosaccharide comprising contacting a reaction mixture comprising anactivated GlcNAc and an acceptor in the presence of a polypeptide of theinvention.

[0196] Examples of acceptors for use in the method for preparing anoligosaccharide are a saccharide, oligosaccharides, polysaccharides,glycopeptides, glycopolypeptides, or glycolipids which are eithersynthetic with linkers at the reducing end or naturally occurringstructures, for example, asialo-agalacto-fetuin glycopeptide. Theactivated GlcNAc may be part of a nucleotide-sugar, adolichol-phosphate-sugar, or dolichol-pyrophosphate-oligosaccharide.

[0197] In an embodiment of the invention, the otigosaccharides areprepared on a carrier that is non-toxic to a mammal, in particular ahuman, such as a lipid isoprenoid or polyisoprenoid alcohol. An exampleof a suitable carrier is dolichol phosphate. The oligosaccharide may beattached to a carrier via a labile bond allowing for chemical removal ofthe oligosaccharide from the lipid carrier. In the alternative, theoligosaccharide transferase may be used to transfer the oligosaccharidefrom a lipid carrier to a polypeptide.

[0198] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLE 1

[0199] A cDNA sequence of a human Core2b GlcNAc-T homolog (accessionnumber AA397800) was identified by similarity matching using theGeneBank ESTdatabase. This EST cDNA clone was sequenced (937 base pairs)and when translatedwas shown to be 60% similar (47% identical) to the 3′end of the human Core2 amino acid sequence. This information initiated asearch for the entire sequence of this human Core2-like cDNA using twodifferent methods; screening a human colon cDNA library by colony plaquelifts and 5′ RACE (rapid amplification of cDNA ends).

[0200] A human colon cDNA library (Uni-ZAP XR library (Stratagene Cat #937221) was screened (using standard protocols) with a ³²P-dCTP labeled485 base pair cDNA probe generated by restriction enzyme digestions ofthe Core2b GlcNAc-T EST cDNA with Kpn1 and EcoR1. Two and a half millionphage clones were screened and 2 positive clones were identified. Eachclone was purified to homogeneity by three subsequent rounds ofscreening. In vivo excision of the pBluescript phagmids of each clonewas isolated using conventional methods from Stratagene. The cDNA insertof one clone (clone 6₄₋₄) was excised by EcoR1 and Xho1 digestion andfound to be 1350 base pairs in length. This clone was sequenced using T3and T7 primers and is 73% similar (56% identical) to the 3′ end of humanCore2. The cDNA insert of the other clone (clone 30₇) was also sequencedand determined to be 1496 base pairs in length and is a 5′ extension ofclone 6₄₋₄. Clone 30₇ wasdesignated as hCore2b GlcNAc-T and found to be75% similar (59% identical) to human Core2.

[0201] The 5′ RACE protocol was used to isolate the 5′ end of thehCore2b GlcNAc-T cDNA sequence. First strand cDNA synthesis wasperformed using a PCR primer that was incubated [(primer RACEC2bGlcNAc-T3-3B-CGCCTCTTTGAAAGTTTCTGGG (SEQ. ID. NO. 15)] (100 mMfinalconcentration) with 2 μg of mRNA from normal human colon tissue andincubated for 10 minutes at 85° C. and then chilled on ice for 1 minute.To this mixture was added, to final concentrations, 20 mM Tris-HCl (pH8.4), 50 mM KCl, 2.5 mM MgCl₂, 10 mM DTT, 400 μM each dATP, dCTP, dGTP,dTTP and 200 Units of Superscript II RT (GIBCO-BRL) and incubated for 50minutes at 42° C. The reaction was terminated by placing it at 70° C.for 15 minutes which was then incubated with 2 Units of RNAse andincubated for an additional 30 minutes. The generated cDNA was purifiedby using GlassMax DNA spin cartridges following the manufacturer'sinstructions (GIBCO-BRL). The isolated cDNA was tailed with terminaldeoxynucleotidyl transferase (TdT) that added homopolymeric dCTP tailsto the 3′ ends of the cDNA in a reaction that was incubated for 10minutes at 37° C. with a final composition of 10 mM Tris-HCl (pH 8.4),25 mM KCl, 1.5 mM MgCl₂, 200 μM dCTP and 1 Unit of TdT. The TdT was heatinactivated for 10 minutes at 65° C. The tailed cDNA (5 μl) wasamplified by PCR using two primers [primer RACEC2bGlcNAc-T3-1B-CTCACAGTCTCTGGTGAGGGAG (SEQ. ID. NO. 16)] and an AbridgedAnchor primer sequence not provided from GIBCO-BRL) with the finalcomposition of the reaction as 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5mM MgCl₃400 mM primer RACEC2b GlcNAc-T3-1B, 400 mM Abridged Anchorprimer, 200 μM each dATP, dCTP, dGTP, dTTP and 2.5 Units of Taq DNApolymerase. This reaction was transferred to a thermal cyclerpre-equilibrated to 94° C. Thirty five cycles of PCR was performed withthe following cycling protocol: pre-denaturation at 94° C. for 4minutes, denaturation at 94° C. for 1 minute, annealing of primers at62° C. for 2 minutes, primer extension at 72° C. for 2 minutes and finalextension at 72° C. for 10 minutes. The 5′ RACE products were analyzedusing standard agarose gel electrophoresis protocols. No visible bandswere observed, therefore a Southern blot of these products was performedusing the identical hCore2b GlcNAc-T probe used for screening. TheSouthern blot revealed that an approximately 800 bp band specificallyhybridized to the probe. Another gel of the 5′ RACE products was runandthe region between 1000 and 400 base pairs was isolated using a DNAgel extraction kit from Stratagene and subcloned into the T/A Bluescriptvector using standard procedures. Several cDNA fragments were subclonedinto the Bluescript vector and were sequenced. One particular clone (537base pairs) was found to represent the 5′ end of hCore2b GlcNAc-T. Theentire Core2b ClcNAc-T CDNA is 438 amino acids in length and is 57%identical and 72% similar to human Core2. The mouse core 2b genesequence was isolated and sequenced during the preparation of a core 2bknock out mouse. The mouse nucleic acid sequence is SEQ ID NO. 19, andthe deduced mouse amino acid sequence is SEQ ID NO. 20.

EXAMPLE 2 Expression of Core2b GlcNAc-T

[0202] Northern Blot Analysis of Human Tissues

[0203] Human multiple tissue and tumor cell line Northern blots wereobtained from Clontech. The Northern blot containing mRNA from humanstomach and liver cancer tissues as well as normal tissues was obtainedfrom Invitrogen. All Northern blots contained 2 μg of mRNA/lane. Theseblots were hybridized with [α-³²P]dCTP-labeled Kpnl/Egrl fragment frombp 310 to 766. Amersham multiprime DNA labeling kit and [α-³²P]dCTP(3000 Ci/mol) were used for labeling. Northern blots were hybridizedunder stringent conditions following the recommended protocol (Clontech)and exposed to x-ray film or phosphoimager.

[0204] Results

[0205] The expression pattern of core2b GlcNAc-T was examined indifferent human tissues. Hybridization of Core2b GlcNAc-T cDNA probe toNorthern blots under stringent conditions revealed the presence of core2b GlcNAc-T mRNA in colon, kidney, and small intestine (FIG. 3). Furtheranalysis showed wide expression of core 2b GlcNAc-T mRNA throughouthuman gastrointestinal tissues (FIG. 4). Northern blots from tumor andnormal tissues indicated overexpression of core 2b GlcNAc-T mRNA intumors from liver and stomach when compared to normal tissues (FIG. 5).

EXAMPLE 3 Quantitative PCR of hcore2b

[0206] RNA was extracted from each sample (normal and cancerous humancolon tissue) using a standard RNA extraction method. Carefulquantitation of the extracted RNA was performed. First-strand cDNAsynthesis was generated using 5 μg of RNA from each sample, 1 μg ofoligo dT (Pharnacia) and H₂0 to a volume of 15 μl. Each sample washeated to 70° C. for 5 minutes and then chilled on ice. To 15 μl of thisRNA mixture was added 2 μl of 10× PCR buffer (200 mM Tris-HCL (pH 84.),500 mM KCl), 2 μl of dNTP (10 mM), 0.5 μl of Superscript (GIBC-OBRL) and0.5 μl of H₂0. The reaction conditions for cDNA synthesis involved 10minutes at 23° C., 1 hour at 42° C. and 10 minutes at 95° C. Differentvolumes of each cDNA sample (1 to 7 μl) were tested using a PCR reactionwith primers to a housekeeping gene β-Microglobulin (β-MG, Clontech#5438-3). The PCR reaction included the following reagents: 5 μl of 10×PCR buffer (200 mM Tris-HCL (pH 84.), 500 mM KCl), 1.5 μl of 50 mM MgCl,1 μl of 10 mM dNTP, 1 l of each of the β-MG primers, CDNA samples(volumes 1-7 μl), 0.5 μl of Platinum Taq polymerase (5 U/μl-GIBCO-BRL)and H₂0 to a volume of 50 μl. The reaction conditions included 94° C.for 5 minutes followed by 22 cycles of 94° C. for 1 minute, 60° C. for 1minute and 72° C. for 1 minute. A linear curve of the β-MG amplifiedproduct was observed and each sample was then standardized to an equalamount of β-MG PCR product. Using the standardized cDNA concentrations,hcore2b primers [5′TATCACTTTGAGGTAGTGAGAGAC3′/5′TACGTTATAAGACAATCTATGGG3′, (SEQ ID. Nos. 17and 18 respectively)] and the β-MG primers were added to a PCR reactionmixture consisting of 5 μl of 10× PCR buffer (200 mM Tris-HCL (pH 84.),500 mM KCl), 1.5 μl of 50 mM MgCl, 1 μl of 10 mM dNTP, CDNA samples(volumes 3-6 μl depending on each sample ), 0.5 μl of Platinum Taqpolymerase (5 U/μl-GIBCO-BRL) and H₂O to a volume of 50 μ1. The PCRconditions included 94° C. for 5 minutes followed by 22 cycles of 94° C.for 1 minute, 60° C. for 1 minute and 72° C. for 1 minute. Tenmicroliters of each PCR sample was run out on an agarose gel foranalysis. Core 2b GlcNAc-T mRNA was found in colon cancer and livermetastasis samples (FIG. 6).

[0207] The present invention is not to be limited in scope by thespecific embodiments described herein, since such embodiments areintended as but single illustrations of one aspect of the invention andany functionally equivalent embodiments are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

[0208] All publications, patents and patent applications referred toherein are incorporated by reference in their entirety to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety. All publications, patents and patentapplications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the cell lines, vectors,methodologies etc. which are reported therein which might be used inconnection with the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

[0209] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

1 20 1 1317 DNA Artificial Sequence Description of Artificial SequenceRecombinant DNA 1 atggttcaat ggaagagact ctgccagctg cattacttgt gggctctgggctgctatatg 60 ctgctggcca ctgtggctct gaaactttct ttcaggttga agtgtgactctgaccacttg 120 ggtctggagt ccagggaatc tcaaagccag tactgtagga atatcttgtataatttcctg 180 aaacttccag caaagaggtc tatcaactgt tcaggggtca cccgaggggaccaagaggca 240 gtgcttcagg ctattctgaa taacctggag gtcaagaaga agcgagagcctttcacagac 300 acccactacc tctccctcac cagagactgt gagcacttca aggctgaaaggaagttcata 360 cagttcccac tgagcaaaga agaggtggag ttccctattg catactctatggtgattcat 420 gagaagattg aaaactttga aaggctactg cgagctgtgt atgcccctcagaacatatac 480 tgtgtccatg tggatgagaa gtccccagaa actttcaaag aggcggtcaaagcaattatt 540 tcttgcttcc caaatgtctt catagccagt aagctggttc gggtggtttatgcctcctgg 600 tccagggtgc aagctgacct caactgcatg gaagacttgc tccagagctcagtgccgtgg 660 aaatacttcc tgaatacatg tgggacggac tttcctataa agagcaatgcagagatggtc 720 caggctctca agatgttgaa tgggaggaat agcatggagt cagaggtacctcctaagcac 780 aaagaaaccc gctggaaata tcactttgag gtagtgagag acacattacacctaaccaac 840 aagaagaagg atcctccccc ttataattta actatgttta cagggaatgcgtacattgtg 900 gcttcccgag atttcgtcca acatgttttg aagaacccta aatcccaacaactgattgaa 960 tgggtaaaag acacttatag cccagatgaa cacctctggg ccacccttcagcgtgcacgg 1020 tggatgcctg gctctgttcc caaccacccc aagtacgaca tctcagacatgacttctatt 1080 gccaggctgg tcaagtggca gggtcatgag ggagacatcg ataagggtgctccttatgct 1140 ccctgctctg gaatccacca gcgggctatc tgcgtttatg gggctggggacttgaattgg 1200 atgcttcaaa accatcacct gttggccaac aagtttgacc caaaggtagatgataatgct 1260 cttcagtgct tagaagaata cctacgttat aaggccatct atgggactgaactttga 1317 2 438 PRT Artificial Sequence Description of ArtificialSequence Recombinant amino acid 2 Met Val Gln Trp Lys Arg Leu Cys GlnLeu His Tyr Leu Trp Ala Leu 1 5 10 15 Gly Cys Tyr Met Leu Leu Ala ThrVal Ala Leu Lys Leu Ser Phe Arg 20 25 30 Leu Lys Cys Asp Ser Asp His LeuGly Leu Glu Ser Arg Glu Ser Gln 35 40 45 Ser Gln Tyr Cys Arg Asn Ile LeuTyr Asn Phe Leu Lys Leu Pro Ala 50 55 60 Lys Arg Ser Ile Asn Cys Ser GlyVal Thr Arg Gly Asp Gln Glu Ala 65 70 75 80 Val Leu Gln Ala Ile Leu AsnAsn Leu Glu Val Lys Lys Lys Arg Glu 85 90 95 Pro Phe Thr Asp Thr His TyrLeu Ser Leu Thr Arg Asp Cys Glu His 100 105 110 Phe Lys Ala Glu Arg LysPhe Ile Gln Phe Pro Leu Ser Lys Glu Glu 115 120 125 Val Glu Phe Pro IleAla Tyr Ser Met Val Ile His Glu Lys Ile Glu 130 135 140 Asn Phe Glu ArgLeu Leu Arg Ala Val Tyr Ala Pro Gln Asn Ile Tyr 145 150 155 160 Cys ValHis Val Asp Glu Lys Ser Pro Glu Thr Phe Lys Glu Ala Val 165 170 175 LysAla Ile Ile Ser Cys Phe Pro Asn Val Phe Ile Ala Ser Lys Leu 180 185 190Val Arg Val Val Tyr Ala Ser Trp Ser Arg Val Gln Ala Asp Leu Asn 195 200205 Cys Met Glu Asp Leu Leu Gln Ser Ser Val Pro Trp Lys Tyr Phe Leu 210215 220 Asn Thr Cys Gly Thr Asp Phe Pro Ile Lys Ser Asn Ala Glu Met Val225 230 235 240 Gln Ala Leu Lys Met Leu Asn Gly Arg Asn Ser Met Glu SerGlu Val 245 250 255 Pro Pro Lys His Lys Glu Thr Arg Trp Lys Tyr His PheGlu Val Val 260 265 270 Arg Asp Thr Leu His Leu Thr Asn Lys Lys Lys AspPro Pro Pro Tyr 275 280 285 Asn Leu Thr Met Phe Thr Gly Asn Ala Tyr IleVal Ala Ser Arg Asp 290 295 300 Phe Val Gln His Val Leu Lys Asn Pro LysSer Gln Gln Leu Ile Glu 305 310 315 320 Trp Val Lys Asp Thr Tyr Ser ProAsp Glu His Leu Trp Ala Thr Leu 325 330 335 Gln Arg Ala Arg Trp Met ProGly Ser Val Pro Asn His Pro Lys Tyr 340 345 350 Asp Ile Ser Asp Met ThrSer Ile Ala Arg Leu Val Lys Trp Gln Gly 355 360 365 His Glu Gly Asp IleAsp Lys Gly Ala Pro Tyr Ala Pro Cys Ser Gly 370 375 380 Ile His Gln ArgAla Ile Cys Val Tyr Gly Ala Gly Asp Leu Asn Trp 385 390 395 400 Met LeuGln Asn His His Leu Leu Ala Asn Lys Phe Asp Pro Lys Val 405 410 415 AspAsp Asn Ala Leu Gln Cys Leu Glu Glu Tyr Leu Arg Tyr Lys Ala 420 425 430Ile Tyr Gly Thr Glu Leu 435 3 2108 DNA Artificial Sequence Descriptionof Artificial Sequence Recombinant DNA 3 atctgcttcc tggttctatattaaagagga gcctgaaact gttccttgga catcttatga 60 atgtcagaaa ataccttttggagggttaga agatcagggg acatggttgt tcacatttgc 120 tgccacggaa caccgccagtcttcacttgg aaacagaatc acgccttgtg aagagatcat 180 ccctaagcag gagagaagctactaaaggat tgtgtcctcc tccaccttcc ctgtgctcgg 240 tctccacctg tctcccattctgtgacgatg gttcaatgga agagactctg ccagctgcat 300 tacttgtggg ctctgggctgctatatgctg ctggccactg tggctctgaa actttctttc 360 aggttgaagt gtgactctgaccacttgggt ctggagtcca gggaatctca aagccagtac 420 tgtaggaata tcttgtataatttcctgaaa cttccagcaa agaggtctat caactgttca 480 ggggtcaccc gaggggaccaagaggcagtg cttcaggcta ttctgaataa cctggaggtc 540 aagaagaagc gagagcctttcacagacacc cactacctct ccctcaccag agactgtgag 600 cacttcaagg ctgaaaggaagttcatacag ttcccactga gcaaagaaga ggtggagttc 660 cctattgcat actctatggtgattcatgag aagattgaaa actttgaaag gctactgcga 720 gctgtgtatg cccctcagaacatatactgt gtccatgtgg atgagaagtc cccagaaact 780 ttcaaagagg cggtcaaagcaattatttct tgcttcccaa atgtcttcat agccagtaag 840 ctggttcggg tggtttatgcctcctggtcc agggtgcaag ctgacctcaa ctgcatggaa 900 gacttgctcc agagctcagtgccgtggaaa tacttcctga atacatgtgg gacggacttt 960 cctataaaga gcaatgcagagatggtccag gctctcaaga tgttgaatgg gaggaatagc 1020 atggagtcag aggtacctcctaagcacaaa gaaacccgct ggaaatatca ctttgaggta 1080 gtgagagaca cattacacctaaccaacaag aagaaggatc ctccccctta taatttaact 1140 atgtttacag ggaatgcgtacattgtggct tcccgagatt tcgtccaaca tgttttgaag 1200 aaccctaaat cccaacaactgattgaatgg gtaaaagaca cttatagccc agatgaacac 1260 ctctgggcca cccttcagcgtgcacggtgg atgcctggct ctgttcccaa ccaccccaag 1320 tacgacatct cagacatgacttctattgcc aggctggtca agtggcaggg tcatgaggga 1380 gacatcgata agggtgctccttatgctccc tgctctggaa tccaccagcg ggctatctgc 1440 gtttatgggg ctggggacttgaattggatg cttcaaaacc atcacctgtt ggccaacaag 1500 tttgacccaa aggtagatgataatgctctt cagtgcttag aagaatacct acgttataag 1560 gccatctatg ggactgaactttgagacaca ctatgagagc gttgctacct gtggggcaag 1620 agcatgtaca aacatgctcagaacttgctg ggacagtgtg ggtgggagac cagggctttg 1680 caattcgtgg catcctttaggataagaggg ctgctattag attgtgggta agtagatctt 1740 ttgccttgca aattgctgcctgggtgaatg ctgcttgttc tctcacccct aaccctagta 1800 gttcctccac taactttctcactaagtgag aatgagaact gctgtgatag ggagagtgaa 1860 ggagggatat gtggtagagcacttgatttc agttgaatgc ctgctggtag cttttccatt 1920 ctgtggagct gccgttcctaataattccag gtttggtagc gtggaggaga actttgatgg 1980 aaagagaacc ttcccttctgtactgttaac ttaaaaataa atagctcctg attcaaagta 2040 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagggagc 2100 tcgaattc 2108 4 663 PRTArtificial Sequence Description of Artificial Sequence Recombinant aminoacid 4 Ile Cys Phe Leu Val Leu Tyr Arg Gly Ala Asn Cys Ser Leu Asp Ile 15 10 15 Leu Met Ser Glu Asn Thr Phe Trp Arg Val Arg Arg Ser Gly Asp Met20 25 30 Val Val His Ile Cys Cys His Gly Thr Pro Pro Val Phe Thr Trp Lys35 40 45 Gln Asn His Ala Leu Arg Asp His Pro Ala Gly Glu Lys Leu Leu Lys50 55 60 Asp Cys Val Leu Leu His Leu Pro Cys Ala Arg Ser Pro Pro Val Ser65 70 75 80 His Ser Val Thr Met Val Gln Trp Lys Arg Leu Cys Gln Leu HisTyr 85 90 95 Leu Trp Ala Leu Gly Cys Tyr Met Leu Leu Ala Thr Val Ala LeuLys 100 105 110 Leu Ser Phe Arg Leu Lys Cys Asp Ser Asp His Leu Gly LeuGlu Ser 115 120 125 Arg Glu Ser Gln Ser Gln Tyr Cys Arg Asn Ile Leu TyrAsn Phe Leu 130 135 140 Lys Leu Pro Ala Lys Arg Ser Ile Asn Cys Ser GlyVal Thr Arg Gly 145 150 155 160 Asp Gln Glu Ala Val Leu Gln Ala Ile LeuAsn Asn Leu Glu Val Lys 165 170 175 Lys Lys Arg Glu Pro Phe Thr Asp ThrHis Tyr Leu Ser Leu Thr Arg 180 185 190 Asp Cys Glu His Phe Lys Ala GluArg Lys Phe Ile Gln Phe Pro Leu 195 200 205 Ser Lys Glu Glu Val Glu PhePro Ile Ala Tyr Ser Met Val Ile His 210 215 220 Glu Lys Ile Glu Asn PheGlu Arg Leu Leu Arg Ala Val Tyr Ala Pro 225 230 235 240 Gln Asn Ile TyrCys Val His Val Asp Glu Lys Ser Pro Glu Thr Phe 245 250 255 Lys Glu AlaVal Lys Ala Ile Ile Ser Cys Phe Pro Asn Val Phe Ile 260 265 270 Ala SerLys Leu Val Arg Val Val Tyr Ala Ser Trp Ser Arg Val Gln 275 280 285 AlaAsp Leu Asn Cys Met Glu Asp Leu Leu Gln Ser Ser Val Pro Trp 290 295 300Lys Tyr Phe Leu Asn Thr Cys Gly Thr Asp Phe Pro Ile Lys Ser Asn 305 310315 320 Ala Glu Met Val Gln Ala Leu Lys Met Leu Asn Gly Arg Asn Ser Met325 330 335 Glu Ser Glu Val Pro Pro Lys His Lys Glu Thr Arg Trp Lys TyrHis 340 345 350 Phe Glu Val Val Arg Asp Thr Leu His Leu Thr Asn Lys LysLys Asp 355 360 365 Pro Pro Pro Tyr Asn Leu Thr Met Phe Thr Gly Asn AlaTyr Ile Val 370 375 380 Ala Ser Arg Asp Phe Val Gln His Val Leu Lys AsnPro Lys Ser Gln 385 390 395 400 Gln Leu Ile Glu Trp Val Lys Asp Thr TyrSer Pro Asp Glu His Leu 405 410 415 Trp Ala Thr Leu Gln Arg Ala Arg TrpMet Pro Gly Ser Val Pro Asn 420 425 430 His Pro Lys Tyr Asp Ile Ser AspMet Thr Ser Ile Ala Arg Leu Val 435 440 445 Lys Trp Gln Gly His Glu GlyAsp Ile Asp Lys Gly Ala Pro Tyr Ala 450 455 460 Pro Cys Ser Gly Ile HisGln Arg Ala Ile Cys Val Tyr Gly Ala Gly 465 470 475 480 Asp Leu Asn TrpMet Leu Gln Asn His His Leu Leu Ala Asn Lys Phe 485 490 495 Asp Pro LysVal Asp Asp Asn Ala Leu Gln Cys Leu Glu Glu Tyr Leu 500 505 510 Arg TyrLys Ala Ile Tyr Gly Thr Glu Leu Asp Thr Leu Glu Arg Cys 515 520 525 TyrLeu Trp Gly Lys Ser Met Tyr Lys His Ala Gln Asn Leu Leu Gly 530 535 540Gln Cys Gly Trp Glu Thr Arg Ala Leu Gln Phe Val Ala Ser Phe Arg 545 550555 560 Ile Arg Gly Leu Leu Leu Asp Cys Gly Val Asp Leu Leu Pro Cys Lys565 570 575 Leu Leu Pro Gly Met Leu Leu Val Leu Ser Pro Leu Thr Leu ValVal 580 585 590 Pro Pro Leu Thr Phe Ser Leu Ser Glu Asn Glu Asn Cys CysAsp Arg 595 600 605 Glu Ser Glu Gly Gly Ile Cys Gly Arg Ala Leu Asp PheSer Met Pro 610 615 620 Ala Gly Ser Phe Ser Ile Leu Trp Ser Cys Arg SerPhe Gln Val Trp 625 630 635 640 Arg Gly Gly Glu Leu Trp Lys Glu Asn LeuPro Phe Cys Thr Val Asn 645 650 655 Leu Lys Ile Asn Ser Ser Phe 660 5524 DNA Artificial Sequence Description of Artificial SequenceRecombinant DNA 5 gacacactat gagagcgttg ctacctgtgg ggcaagagca tgtacaaacatgctcagaac 60 ttgctgggac agtgtgggtg ggagaccagg gctttgcaat tcgtggcatcctttaggata 120 agagggctgc tattagattg tgggtaagta gatcttttgc cttgcaaattgctgcctggg 180 tgaatgctgc ttgttctctc acccctaacc ctagtagttc ctccactaactttctcacta 240 agtgagaatg agaactgctg tgatagggag agtgaaggag ggatatgtggtagagcactt 300 gatttcagtt gaatgcctgc tggtagcttt tccattctgt ggagctgccgttcctaataa 360 ttccaggttt ggtagcgtgg aggagaactt tgatggaaag agaaccttcccttctgtact 420 gttaacttaa aaataaatag ctcctgattc aaagtaaaaa aaaaaaaaaaaaaaaaaaaa 480 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gggagctcga attc 524 6267 DNA Artificial Sequence Description of Artificial SequenceRecombinant DNA 6 atctgcttcc tggttctata ttaaagagga gcctgaaact gttccttggacatcttatga 60 atgtcagaaa ataccttttg gagggttaga agatcagggg acatggttgttcacatttgc 120 tgccacggaa caccgccagt cttcacttgg aaacagaatc acgccttgtgaagagatcat 180 ccctaagcag gagagaagct actaaaggat tgtgtcctcc tccaccttccctgtgctcgg 240 tctccacctg tctcccattc tgtgacg 267 7 9 PRT ArtificialSequence Description of Artificial Sequence Recombinant amino acid 7 MetVal Gln Trp Lys Arg Leu Cys Gln 1 5 8 23 PRT Artificial SequenceDescription of Artificial Sequence Recombinant amino acid 8 Leu His TyrLeu Trp Ala Leu Gly Cys Tyr Met Leu Leu Ala Thr Val 1 5 10 15 Ala LeuLys Leu Ser Phe Arg 20 9 406 PRT Artificial Sequence Description ofArtificial Sequence Recombinant amino acid 9 Leu Lys Cys Asp Ser Asp HisLeu Gly Leu Glu Ser Arg Glu Ser Gln 1 5 10 15 Ser Gln Tyr Cys Arg AsnIle Leu Tyr Asn Phe Leu Lys Leu Pro Ala 20 25 30 Lys Arg Ser Ile Asn CysSer Gly Val Thr Arg Gly Asp Gln Glu Ala 35 40 45 Val Leu Gln Ala Ile LeuAsn Asn Leu Glu Val Lys Lys Lys Arg Glu 50 55 60 Pro Phe Thr Asp Thr HisTyr Leu Ser Leu Thr Arg Asp Cys Glu His 65 70 75 80 Phe Lys Ala Glu ArgLys Phe Ile Gln Phe Pro Leu Ser Lys Glu Glu 85 90 95 Val Glu Phe Pro IleAla Tyr Ser Met Val Ile His Glu Lys Ile Glu 100 105 110 Asn Phe Glu ArgLeu Leu Arg Ala Val Tyr Ala Pro Gln Asn Ile Tyr 115 120 125 Cys Val HisVal Asp Glu Lys Ser Pro Glu Thr Phe Lys Glu Ala Val 130 135 140 Lys AlaIle Ile Ser Cys Phe Pro Asn Val Phe Ile Ala Ser Lys Leu 145 150 155 160Val Arg Val Val Tyr Ala Ser Trp Ser Arg Val Gln Ala Asp Leu Asn 165 170175 Cys Met Glu Asp Leu Leu Gln Ser Ser Val Pro Trp Lys Tyr Phe Leu 180185 190 Asn Thr Cys Gly Thr Asp Phe Pro Ile Lys Ser Asn Ala Glu Met Val195 200 205 Gln Ala Leu Lys Met Leu Asn Gly Arg Asn Ser Met Glu Ser GluVal 210 215 220 Pro Pro Lys His Lys Glu Thr Arg Trp Lys Tyr His Phe GluVal Val 225 230 235 240 Arg Asp Thr Leu His Leu Thr Asn Lys Lys Lys AspPro Pro Pro Tyr 245 250 255 Asn Leu Thr Met Phe Thr Gly Asn Ala Tyr IleVal Ala Ser Arg Asp 260 265 270 Phe Val Gln His Val Leu Lys Asn Pro LysSer Gln Gln Leu Ile Glu 275 280 285 Trp Val Lys Asp Thr Tyr Ser Pro AspGlu His Leu Trp Ala Thr Leu 290 295 300 Gln Arg Ala Arg Trp Met Pro GlySer Val Pro Asn His Pro Lys Tyr 305 310 315 320 Asp Ile Ser Asp Met ThrSer Ile Ala Arg Leu Val Lys Trp Gln Gly 325 330 335 His Glu Gly Asp IleAsp Lys Gly Ala Pro Tyr Ala Pro Cys Ser Gly 340 345 350 Ile His Gln ArgAla Ile Cys Val Tyr Gly Ala Gly Asp Leu Asn Trp 355 360 365 Met Leu GlnAsn His His Leu Leu Ala Asn Lys Phe Asp Pro Lys Val 370 375 380 Asp AspAsn Ala Leu Gln Cys Leu Glu Glu Tyr Leu Arg Tyr Lys Ala 385 390 395 400Ile Tyr Gly Thr Glu Leu 405 10 27 DNA Artificial Sequence Description ofArtificial Sequence Recombinant DNA 10 atggttcaat ggaagagact ctgccag 2711 69 DNA Artificial Sequence Description of Artificial SequenceRecombinant DNA 11 ctgcattact tgtgggctct gggctgctat atgctgctggccactgtggc tctgaaactt 60 tctttcagg 69 12 1221 DNA Artificial SequenceDescription of Artificial Sequence Recombinant DNA 12 ttgaagtgtgactctgacca cttgggtctg gagtccaggg aatctcaaag ccagtactgt 60 aggaatatcttgtataattt cctgaaactt ccagcaaaga ggtctatcaa ctgttcaggg 120 gtcacccgaggggaccaaga ggcagtgctt caggctattc tgaataacct ggaggtcaag 180 aagaagcgagagcctttcac agacacccac tacctctccc tcaccagaga ctgtgagcac 240 ttcaaggctgaaaggaagtt catacagttc ccactgagca aagaagaggt ggagttccct 300 attgcatactctatggtgat tcatgagaag attgaaaact ttgaaaggct actgcgagct 360 gtgtatgcccctcagaacat atactgtgtc catgtggatg agaagtcccc agaaactttc 420 aaagaggcggtcaaagcaat tatttcttgc ttcccaaatg tcttcatagc cagtaagctg 480 gttcgggtggtttatgcctc ctggtccagg gtgcaagctg acctcaactg catggaagac 540 ttgctccagagctcagtgcc gtggaaatac ttcctgaata catgtgggac ggactttcct 600 ataaagagcaatgcagagat ggtccaggct ctcaagatgt tgaatgggag gaatagcatg 660 gagtcagaggtacctcctaa gcacaaagaa acccgctgga aatatcactt tgaggtagtg 720 agagacacattacacctaac caacaagaag aaggatcctc ccccttataa tttaactatg 780 tttacagggaatgcgtacat tgtggcttcc cgagatttcg tccaacatgt tttgaagaac 840 cctaaatcccaacaactgat tgaatgggta aaagacactt atagcccaga tgaacacctc 900 tgggccacccttcagcgtgc acggtggatg cctggctctg ttcccaacca ccccaagtac 960 gacatctcagacatgacttc tattgccagg ctggtcaagt ggcagggtca tgagggagac 1020 atcgataagggtgctcctta tgctccctgc tctggaatcc accagcgggc tatctgcgtt 1080 tatggggctggggacttgaa ttggatgctt caaaaccatc acctgttggc caacaagttt 1140 gacccaaaggtagatgataa tgctcttcag tgcttagaag aatacctacg ttataaggcc 1200 atctatgggactgaactttg a 1221 13 2109 DNA Homo sapiens 13 gtgaagtgct cagaatggggcaggatgtca cctggaatca gcactaagtg attcagactt 60 tccttacttt taaatgtgctgctcttcatt tcaagatgcc gttgcagctc tgataaatgc 120 aaactgacaa ccttcaaggccacgacggag ggaaaatcat tggtgcttgg agcatagaag 180 actgcccttc acaaaggaaatccctgatta ttgtttgaaa tgctgaggac gttgctgcga 240 aggagacttt tttcttatcccaccaaatac tactttatgg ttcttgtttt atccctaatc 300 accttctccg ttttaaggattcatcaaaag cctgaatttg taagtgtcag acacttggag 360 cttgctgggg agaatcctagtagtgatatt aattgcacca aagttttaca gggtgatgta 420 aatgaaatcc aaaaggtaaagcttgagatc ctaacagtga aatttaaaaa gcgccctcgg 480 tggacacctg acgactatataaacatgacc agtgactgtt cttctttcat caagagacgc 540 aaatatattg tagaaccccttagtaaagaa gaggcggagt ttccaatagc atattctata 600 gtggttcatc acaagattgaaatgcttgac aggctgctga gggccatcta tatgcctcag 660 aatttctatt gcgttcatgtggacacaaaa tccgaggatt cctatttagc tgcagtgatg 720 ggcatcgctt cctgttttagtaatgtcttt gtggccagcc gattggagag tgtggtttat 780 gcatcgtgga gccgggttcaggctgacctc aactgcatga aggatctcta tgcaatgagt 840 gcaaactgga agtacttgataaatctttgt ggtatggatt ttcccattaa aaccaaccta 900 gaaattgtca ggaagctcaagttgttaatg ggagaaaaca acctggaaac ggagaggatg 960 ccatcccata aagaagaaaggtggaagaag cggtatgagg tcgttaatgg aaagctgaca 1020 aacacaggga ctgtcaaaatgcttcctcca ctcgaaacac ctctcttttc tggcagtgcc 1080 tacttcgtgg tcagtagggagtatgtgggg tatgtactac agaatgaaaa aatccaaaag 1140 ttgatggagt gggcacaagacacatacagc cctgatgagt atctctgggc caccatccaa 1200 aggattcctg aagtcccgggctcactccct gccagccata agtatgatct atctgacatg 1260 caagcagttg ccaggtttgtcaagtggcag tactttgagg gtgatgtttc caagggtgct 1320 ccctacccgc cctgcgatggagtccatgtg cgctcagtgt gcattttcgg agctggtgac 1380 tgaactggat gctgcgcaaacaccacttgt ttgccaataa gtttgacgtg gatgttgacc 1440 tctttgccat ccagtgtttggatgagcatt tgagacacaa agctttggag acattaaaac 1500 actgaccatt acgggcaattttatgaacaa gaagaaggat acacaaaacg taccttatct 1560 gtttcccctt ccttgtcagcgtcgggaaga tggtatgaag tcctctttgg ggcagggact 1620 ctagtagatc ttcttgtcagagaagctgca tggtttctgc agagcacagt tagctagaaa 1680 ggtgatagca ttaaatgttcatctagagtt aatagtggga ggagtaaagg tagccttgag 1740 gccagagcag gtagcaaggcattgtggaaa gaggggacca gggtggctgg ggaagaggcc 1800 gatgcataaa gtcagcctgttccaagtgct cagggactta gcaaaatgag aagatgtgac 1860 ctgtgccaaa actattttgagaattttaaa tgtgaccatt tttctggtat gccaataaad 1920 dcttacagca acaaataatcaaagatacaa ttaatctgat attatatttg ttgaaataga 1980 aatttgattg tactataaatgatttttgta aataatttat attctgctct aatactgtac 2040 tgtgtagtgt gtctccgtatgtcatctcag ggagcttaaa atgggcttga tttaacattg 2100 aaaaaaaat 2109 14 428PRT Homo sapiens 14 Met Leu Arg Thr Leu Leu Arg Arg Arg Leu Phe Ser TyrPro Thr Lys 1 5 10 15 Tyr Tyr Phe Met Val Leu Val Leu Ser Leu Ile ThrPhe Ser Val Leu 20 25 30 Arg Ile His Gln Lys Pro Glu Phe Val Ser Val ArgHis Leu Glu Leu 35 40 45 Ala Gly Glu Asn Pro Ser Ser Asp Ile Asn Cys ThrLys Val Leu Gln 50 55 60 Gly Asp Val Asn Glu Ile Gln Lys Val Lys Leu GluIle Leu Thr Val 65 70 75 80 Lys Phe Lys Lys Arg Pro Arg Trp Thr Pro AspAsp Tyr Ile Asn Met 85 90 95 Thr Ser Asp Cys Ser Ser Phe Ile Lys Arg ArgLys Tyr Ile Val Glu 100 105 110 Pro Leu Ser Lys Glu Glu Ala Glu Phe ProIle Ala Tyr Ser Ile Val 115 120 125 Val His His Lys Ile Glu Met Leu AspArg Leu Leu Arg Ala Ile Tyr 130 135 140 Met Pro Gln Asn Phe Tyr Cys ValHis Val Asp Thr Lys Ser Glu Asp 145 150 155 160 Ser Tyr Leu Ala Ala ValMet Gly Ile Ala Ser Cys Phe Ser Asn Val 165 170 175 Phe Val Ala Ser ArgLeu Glu Ser Val Val Tyr Ala Ser Trp Ser Arg 180 185 190 Val Gln Ala AspLeu Asn Cys Met Lys Asp Leu Tyr Ala Met Ser Ala 195 200 205 Asn Trp LysTyr Leu Ile Asn Leu Cys Gly Met Asp Phe Pro Ile Lys 210 215 220 Thr AsnLeu Glu Ile Val Arg Lys Leu Lys Leu Leu Met Gly Glu Asn 225 230 235 240Asn Leu Glu Thr Glu Arg Met Pro Ser His Lys Glu Glu Arg Trp Lys 245 250255 Lys Arg Tyr Glu Val Val Asn Gly Lys Leu Thr Asn Thr Gly Thr Val 260265 270 Lys Met Leu Pro Pro Leu Glu Thr Pro Leu Phe Ser Gly Ser Ala Tyr275 280 285 Phe Val Val Ser Arg Glu Tyr Val Gly Tyr Val Leu Gln Asn GluLys 290 295 300 Ile Gln Lys Leu Met Glu Trp Ala Gln Asp Thr Tyr Ser ProAsp Glu 305 310 315 320 Tyr Leu Trp Ala Thr Ile Gln Arg Ile Pro Glu ValPro Gly Ser Leu 325 330 335 Pro Ala Ser His Lys Tyr Asp Leu Ser Asp MetGln Ala Val Ala Arg 340 345 350 Phe Val Lys Trp Gln Tyr Phe Glu Gly AspVal Ser Lys Gly Ala Pro 355 360 365 Tyr Pro Pro Cys Asp Gly Val His ValArg Ser Val Cys Ile Phe Gly 370 375 380 Ala Gly Asp Leu Asn Trp Met LeuArg Lys His His Leu Phe Ala Asn 385 390 395 400 Lys Phe Asp Val Asp ValAsp Leu Phe Ala Ile Gln Cys Leu Asp Glu 405 410 415 His Leu Arg His LysAla Leu Glu Thr Leu Lys His 420 425 15 22 DNA Artificial SequenceDescription of Artificial Sequence Primer 15 cgcctctttg aaagtttctg gg 2216 22 DNA Artificial Sequence Description of Artificial Sequence Primer16 ctcacagtct ctggtgaggg ag 22 17 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 17 tatcactttg aggtagtgag agac 24 18 23 DNAArtificial Sequence Description of Artificial Sequence Primer 18tacgttataa gacaatctat ggg 23 19 1314 DNA Murine sp. 19 atgacttcctggcagaggct ctgctggcac tatcgcctgt ggaccctggg ttgctacatg 60 ctactggccatccttgccct gaaactgtcc ctcagactga agtgtgactt cgatgccatg 120 gatctggactctgaggaatt tcaaagccag tactgcaggg atctcctgta caagaccctg 180 aagctgccagccaagagttc catcaactgc tcaggggtca ttcgagggga gcagaaagcg 240 gtgacccaggctctgctgaa taacctggaa attaagaaga agcagcagct cttcacagag 300 gccgactaccttaggatgac agcagactgt gagcacttca agaccaagag gaagtttata 360 caggtcccactgagcaagga agaggccagc ttccccattg cgtactccat ggtggtgcat 420 gagaagattgagaacttcga aaggttgctg cgagctgtgt acacccctca gaatgtatac 480 tgtgtccacatggatcagaa gtcttcagaa ccctttaagc aggcagtcag ggccatcgtg 540 tcatgcttccccaatgtctt catagctagt aagttggtgt cagtggtcta tgcttcctgg 600 tccagggtgcaggctgacct aaactgcatg gaagacttgc ttcagagccc cgtgccatgg 660 aaatacctcctgaacacctg tgggacagac tttcccatca aaaccaatgc tgagatggtc 720 aaggccctcaagctattgaa agggcagaac agtatggagt cagaggtacc acctccacat 780 aaaaaatcccgctggaaata tcactatgag gtgacagaca cattgcacat gaccagcaag 840 aggaagacgccgccacctaa taacctaacc atgttcactg ggaatgccta catggtggct 900 tctcgagacttcattgaaca cgtgttcagt aactcaaaag cccggcaact gatcgagtgg 960 gtaaaagacacctatagtcc cgatgagcac ctttgggcca ccctccagcg tgcctcgtgg 1020 atgcctgggtcagatccctt gcatcgaaaa tttgacctgt cagacatgag agccattgcg 1080 agactaaccaagtggtacga ccatgaggga gacattgaga acggggcacc ttacacgtct 1140 tgctcaggaatccaccagcg ggctgtctgt gtttatgggt caggggacct gcactggata 1200 cttcagaaccatcacctctt ggccaacaag tttgacccaa aggtggatga taatgttctt 1260 cagtgtttagaagaatattt acgtcacaaa gccatctatg ggactgaact atga 1314 20 437 PRT Murinesp. 20 Met Thr Ser Trp Gln Arg Leu Cys Trp His Tyr Arg Leu Trp Thr Leu 15 10 15 Gly Cys Tyr Met Leu Leu Ala Ile Leu Ala Leu Lys Leu Ser Leu Arg20 25 30 Leu Lys Cys Asp Phe Asp Ala Met Asp Leu Asp Ser Glu Glu Phe Gln35 40 45 Ser Gln Tyr Cys Arg Asp Leu Leu Tyr Lys Thr Leu Lys Leu Pro Ala50 55 60 Lys Ser Ser Ile Asn Cys Ser Gly Val Ile Arg Gly Glu Gln Lys Ala65 70 75 80 Val Thr Gln Ala Leu Leu Asn Asn Leu Glu Ile Lys Lys Lys GlnGln 85 90 95 Leu Phe Thr Glu Ala Asp Tyr Leu Arg Met Thr Ala Asp Cys GluHis 100 105 110 Phe Lys Thr Lys Arg Lys Phe Ile Gln Val Pro Leu Ser LysGlu Glu 115 120 125 Ala Ser Phe Pro Ile Ala Tyr Ser Met Val Val His GluLys Ile Glu 130 135 140 Asn Phe Glu Arg Leu Leu Arg Ala Val Tyr Thr ProGln Asn Val Tyr 145 150 155 160 Cys Val His Met Asp Gln Lys Ser Ser GluPro Phe Lys Gln Ala Val 165 170 175 Arg Ala Ile Val Ser Cys Phe Pro AsnVal Phe Ile Ala Ser Lys Leu 180 185 190 Val Ser Val Val Tyr Ala Ser TrpSer Arg Val Gln Ala Asp Leu Asn 195 200 205 Cys Met Glu Asp Leu Leu GlnSer Pro Val Pro Trp Lys Tyr Leu Leu 210 215 220 Asn Thr Cys Gly Thr AspPhe Pro Ile Lys Thr Asn Ala Glu Met Val 225 230 235 240 Lys Ala Leu LysLeu Leu Lys Gly Gln Asn Ser Met Glu Ser Glu Val 245 250 255 Pro Pro ProHis Lys Lys Ser Arg Trp Lys Tyr His Tyr Glu Val Thr 260 265 270 Asp ThrLeu His Met Thr Ser Lys Arg Lys Thr Pro Pro Pro Asn Asn 275 280 285 LeuThr Met Phe Thr Gly Asn Ala Tyr Met Val Ala Ser Arg Asp Phe 290 295 300Ile Glu His Val Phe Ser Asn Ser Lys Ala Arg Gln Leu Ile Glu Trp 305 310315 320 Val Lys Asp Thr Tyr Ser Pro Asp Glu His Leu Trp Ala Thr Leu Gln325 330 335 Arg Ala Ser Trp Met Pro Gly Ser Asp Pro Leu His Arg Lys PheAsp 340 345 350 Leu Ser Asp Met Arg Ala Ile Ala Arg Leu Thr Lys Trp TyrAsp His 355 360 365 Glu Gly Asp Ile Glu Asn Gly Ala Pro Tyr Thr Ser CysSer Gly Ile 370 375 380 His Gln Arg Ala Val Cys Val Tyr Gly Ser Gly AspLeu His Trp Ile 385 390 395 400 Leu Gln Asn His His Leu Leu Ala Asn LysPhe Asp Pro Lys Val Asp 405 410 415 Asp Asn Val Leu Gln Cys Leu Glu GluTyr Leu Arg His Lys Ala Ile 420 425 430 Tyr Gly Thr Glu Leu 435

We claim:
 1. An isolated nucleic acid molecule which encodes a core 2bβ-1,6-N-acetylglycosaminyltransferase.
 2. An isolated nucleic acidmolecule as claimed in claim 1 comprising at least 30 nucleotides whichhybridizes to SEQ ID NO. 1, 3, or 12 or the complement of SEQ ID NO. 1,3, or 12 under stringent hybridization conditions.
 3. An isolatednucleic acid molecule as claimed in claim 1 which comprises: (i) anucleic acid sequence encoding a polypeptide having substantial sequenceidentity with an amino acid sequence of SEQ. ID. NO. 2, 4, 7, 8, or 9;(ii) a nucleic acid sequence complementary to (i); (iii) a nucleic acidsequence differing from any of the nucleic acid sequences of (i) or (ii)in codon sequences due to the degeneracy ofthe genetic code; (iv) anucleic acid sequence comprising at least 10 nucleotides capable ofhybridizing under stringent conditions to a nucleic acid sequence ofSEQ. ID. NO. 1, 3, or 12 or to a degenerate form thereof; (v) a nucleicacid sequence encoding a truncation, an analog, an allelic or speciesvariation of a polypeptide comprising an amino acid sequence of SEQ. ID.NO. 2, 4, 7, 8, or 9; or (vi) a fragment, or allelic or speciesvariation of (i), (ii) or (iii).
 4. An isolated nucleic acid molecule asclaimed in claim i which comprises: (i) a nucleic acid sequence havingsubstantial sequence identity with a nucleotide sequence of SEQ. ID. NO.1, 3, 10, 11, or 12; (ii) a nucleic acid sequence complementary to (i);(iii) a nucleic acid sequence differing from any of the nucleic acidsequences of (i) to (ii) in codon sequences due to the degeneracy of thegenetic code; or (iv) a fragment, or allelic or species variation of(i), (ii) or (iii).
 5. An isolated nucleic acid molecule as claimed inclaim 1 which consists essentially of the nucleic acid sequence of SEQID NO. 1 or
 3. 6. A vector comprising a nucleic acid molecule as claimedin claim
 2. 7. A host cell comprising a nucleic acid molecule as claimedin claim
 2. 8. An isolated core 2b β-1,6-N-acetylglycosaminyltransferasepolypeptide comprising the amino acid sequence of SEQ ID NO. 2, or anaturally occurring variant or fragment thereof having mouse core 2bβ-1,6-N-acetylglycosaminyltransferase activity.
 9. An isolated- core 2bβ-1,6-N-acetylglycosaminyltransferase polypeptide comprising an aminoacid sequence of SEQ. ID. NO. 2, 4, 7, 8, or
 9. 10. An isolatedpolypeptide as claimed in claim 9 having at least 58% amino acidsequence identity to an amino acid sequence of SEQ. ID. NO.
 9. 11. Amethod for preparing a core 2b β-1,6-N-acetylglycosaminyltransferasecomprising: (a) transferring a vector as claimed in claim 6 into a hostcell; (b) selecting transformed host cells from untransformed hostcells; (c) culturing a selected transformed host cell under conditionswhich allow expression of the core 2bβ-1,6-N-acetylglycosaminyltransferase; and (d) isolating the core 2bβ-1,6-N-acetylglycosaminyltransferase.
 12. An antibody havingspecificity against an epitope of a polypeptide as claimed in claim 9.13. A probe comprising a sequence encoding a polypeptide as claimed inclaim 9, or a part thereof.
 14. A method of diagnosing and monitoringconditions mediated by a core 2b β-1,6-N-acetylglycosaminyltransferaseby determining the presence of a nucleic acid molecule as claimed inclaim
 2. 15. A diagnostic or prognostic method for colon cancer or livermetastasis in a subject comprising detecting a nucleic acid molecule asclaimed in claim 1 in a sample from the subject.
 16. A method ofdiagnosing and monitoring conditions mediated by a core 2bβ-1,6-N-acetylglycosaminyltransferase by determining the presence of apolypeptide as claimed in claim
 9. 17. A method for screening a compoundfor effectiveness as an antagonist of a polypeptide as claimed in claim9, comprising the steps of a) contacting a sample containing the proteinwith a compound, under conditions wherein antagonist activity of thepolypeptide can be detected, and b) detecting antagonist activity in thesample.
 18. A compound identified by the method of claim
 17. 19. Amethod for detecting a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence of SEQ. ID. NO. 2, 4, 7, 8, or 9 in abiological sample comprising the steps of: (a) hybridizing the nucleicacid molecule of claim 3 to nucleic acids of the biological sample,thereby forming a hybridization complex; and (b) detecting thehybridization complex wherein the presence of the hybridization complexcorrelates with the presence of a nucleic acid molecule encoding thepolypeptide in the biological sample.
 20. A method for treating acondition mediated by a core 2b β-1,6-N-acetylglycosaminyltransferasecomprising administering an effective amount of an antibody as claimedin claim
 12. 21. A gene-based therapy directed at the gastrointestinaltract comprising a polynucleotide comprising all or a portion of aregulatory sequence of SEQ. ID. NO. 5 or 6.